CN101287828A - Discovery, cloning and purification of thermococcus sp. (strain 9 deg n-7) DNA ligase - Google Patents
Discovery, cloning and purification of thermococcus sp. (strain 9 deg n-7) DNA ligase Download PDFInfo
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Abstract
Compositions that describe a thermostable DNA ligase isolated from Thermococcus sp. (strain 9 DEG N-7) and methods for making und using the same are described. The thermostable DMA ligase depends on ATP and not NAD+ as a cofactor during ligation, and retains activity at 100 DEG C.
Description
Technical background
[001] thermophilic Coccus (Thermococcus) is a genus of archeobacteria door (Archaea).Grow in the multiple environment of these ancient biologies under comprising the pyritous extreme condition.It is very limited cultivating these biological abilities in the laboratory, so that rarely known by the people about their biological chemistry or their metabolism.
[002] ligase enzyme is the enzyme that the catalysis phosphodiester bond forms on the strand otch site in duplex DNA.Ligase enzyme is the covalent linkage of catalysis duplex DNA also, generally is flat terminal to flat terminal, and perhaps one is glued terminal sticking terminal to another.Ligase enzyme is cloned from various bacteria, comprises a kind of thermally-stabilised ligase enzyme---thermus aquaticus (Thermus aquaticus) (Taq ligase enzyme).This ligase enzyme is at United States Patent (USP) the 6th, 054, describes in No. 564.
[003] only some thermophilic coccuses are separated, the characteristic of the ligase enzyme that may contain about them or the gene of this proteinoid of encoding know little about it (referring to, Nakatani etc. for example, J.Bacteriology 182:6424-6433 (2000)).Never the ligase enzyme that obtains is for example at United States Patent (USP) the 5th for generic archeobacteria---hot-bulb Pseudomonas (pyrococcus)---, 506,137 and 5,700, No. 672 and in J.Bacteriology 187:6902-6908 (2005) such as Keppetipola, be described.
[004] ligase enzyme has been used to comprise the detection of DNA cloning, order-checking and single nucleotide polymorphism in molecular biological many technology.It is required seeking ligase enzyme at high temperature stable and that have rapid kinetics and a strict specific improvement always.
Summary of the invention
[005] in one embodiment of the present invention, provide to have the active pure basically recombinant protein of dna ligase, wherein this protein and SEQ ID NO:13 have at least 91% amino acid sequence identity.
[006] in further embodiment of the present invention, provide to have the active pure basically protein of dna ligase, wherein dna ligase is with being selected from following dna sequence encoding: (a) with the substantially the same sequence of SEQ ID NO:2; (b) with SEQ ID NO:2 complementary sequence basically; (c) under stringent condition, the sequence of hybridizing basically with SEQ ID NO:2; (d) sequence of coding SEQ ID NO:13.
That [007] points out in the above-described embodiment proteinicly is further characterized in that at incubation under about 100 ℃ temperature after 30 minutes, ligase enzyme has kept at least 25% ligase enzyme activity.And this ligase enzyme is possible to be further characterized in that it uses ATP as cofactor in connection procedure, yet with regard to this purpose, NAD
+Detectable effectiveness is not provided.
[008] in further embodiment, the DNA of coding DNA ligase enzyme is provided, this DNA has and is selected from following sequence: (a) with the substantially the same sequence of SEQ ID NO:2; (b) with SEQ ID NO:2 complementary sequence basically; (c) under stringent condition, the sequence of hybridizing basically with SEQ IDNO:2; (d) sequence of coding SEQ ID NO:13.
[009] in further embodiment, the carrier that contains above-mentioned DNA has been described.In addition, providing can be from the host cell of vector expression ligase enzyme.
[010] in further embodiment, the method that connects phosphodiester bond is provided, this method comprises: the dna ligase of selecting the above-mentioned type; Ligase enzyme is mixed with DNA, and described DNA contains otch at least one chain of this DNA; And connect described phosphodiester bond at described otch.
[011] in an example of this method, dna ligase is from the archeobacteria isolate, is specially the thermally-stabilised ligase enzyme that certain (Thermococcus sp.) (strain 9 deg n-7) of thermophilic Coccus obtains.
The accompanying drawing summary
[012] Fig. 1 a-1 to 1a-5 illustrates the dna sequence dna comparison of 9 ° of N-7DNA ligase enzyme variants (SEQ ID NOS:1-7).
[013] Fig. 1 b-1 to 1b-2 illustrates the protein comparison of certain (strain 9 deg n-7) dna ligase variant (SEQ ID NOS:8-15) of thermophilic Coccus.
[014] Fig. 2 illustrates the plasmid figure that is inserted into certain (strain 9 deg n-7) the dna ligase gene of thermophilic Coccus in the litmus 28i.
[015] Fig. 3 illustrates the plasmid figure that is inserted into certain (strain 9 deg n-7) the dna ligase gene of thermophilic Coccus in the pMalC2x.
[016] Fig. 4 illustrates the protein comparison of thermophilic Coccus certain (strain 9 deg n-7) (SEQ ID NO:15) and the happiness thermophilic coccus of cigarette (Thermococcus fumicolans) (SEQ ID NO:16), the thermophilic coccus of Ke Dakala (Thermococcus kodakaraensis) (SEQ ID NO:17), deep-sea hot-bulb bacterium (Pyrococcus abyssi) (SEQ ID NO:18) and fierce hot-bulb bacterium (pyrococcusfurious) (SEQ ID NO:19).
[017] Fig. 5 illustrates the SDS PAGE of phosphocellurose column component (phosphocellulose column fraction).The following mark of swimming lane:
FT (post circulation) refers to component numbering 23-34;
MW refers to molecular weight standard.
Arrow is pointed out on the gel position with the corresponding band of dna ligase.
[018] Fig. 6 illustrates the thermostability of 9 ° of N-7DNA ligase enzymes.10mM Tris HCl, the 2.5mM MgCl of 30 μ l, pH7.5
2, 2.5mM DTT, 300 μ M ATP and 0.1%TritonX-100---its contain 3 μ l with 1: 100 the dilution pure 9 ° of N-7DNA ligase enzymes, at the 10mM of pH7.5 Tris HCl, 2.5mM MgCl
2,, further continuous three times of dilutions of 2.5mM DTT, 300 μ M ATP and 0.1%Triton X-100.Four groups of identical diluents were 4 ℃, 80 ℃, 90 ℃ or 100 ℃ of following incubations 30 minutes.
[019], sample placed on ice and 10mM Tris HCl, the 2.5mM MgCl of isopyknic pH7.5 for termination reaction
2,, 2.5mM DTT, 300 μ M ATP, 0.1%TritonX-100 and 50 μ g/ml BstEII λ DNA join in every arm.Then 45 ℃ of following incubation reaction things 15 minutes, 50% glycerine, 100mM EDTA and bromjophenol blue with 0.15 volume joins in every arm after this.Then 75 ℃ of following incubation reaction things 5 minutes, and on 1% agarose TBE gel, carry out electrophoresis.
[020] figure A is illustrated in 30 minutes result of incubation on ice.
[021] figure B is illustrated in 30 minutes result of 80 ℃ of following incubations.
[022] figure C is illustrated in 30 minutes result of 90 ℃ of following incubations.
[023] figure D is illustrated in 30 minutes result of 100 ℃ of following incubations.
[024] for each figure, the following appointment of swimming lane:
Not further dilution of swimming lane 1 expression;
[025] in the gel shown in Fig. 7,9 ° of N-7 polysaccharases and Taq polysaccharase in the reparation mixture that contains intestinal bacteria (E.coli) polysaccharase and intestinal bacteria restriction endonuclease IV (Endo IV) relatively.This repairs the DNA incubation of mixture with depurination, and increases.
Embodiment describes in detail
[026] term used herein " thermally-stabilised ligase enzyme (thermostable ligase) " refers to that catalytic dna connects and is keeping its at least 25% active enzyme under 100 ℃, after 30 minutes.Thermostability under extreme temperature is the feature that thermophilic Coccus ligase enzyme (archeobacteria) and thermophilic Pseudomonas (Thermus) ligase enzyme (bacterium) are distinguished.
[027] thermophilic Coccus certain (strain 9 deg n-7) is the kind (PNAS 93:5281 (1996) such as Southworth) from the isolating thermophilic coccus of hydro-thermal spout (hydrothermalvent).
[028] two known immediate relatives (relative) of certain (strain 9 deg n-7) dna ligase of thermophilic Coccus are happiness thermophilic coccus ligase enzyme of cigarette and the thermophilic coccus ligase enzyme of Ke Dakala (JP 2000308494-A/l), and it has 88% and 90% identity respectively at amino acid levels.These two ligase enzymes all are in the news and utilize NAD
+Or ATP is as cofactor, thereby constitutes one group of novel type ligase enzyme.The thermophilic coccus ligase enzyme of happiness cigarette is utilized NAD by report such as Nakatani (J.Bacteriology182:6424-6433 (2000))
+Or ATP is good equally, and the thermophilic coccus ligase enzyme of Ke Dakala is utilizing NAD
+The activity that has the attenuating level when replacing ATP, however thermophilic Coccus certain (strain 9 deg n-7) utilizes NAD
+The time do not have a detectable activity (embodiment 2).
[029] term " substantially the same " and " complementary basically " intend referring to the very big homogeny or the identity of sequence of DNA or aminoacid sequence and evaluation, perhaps very big with complementary sequence homogeny or identity.The sequence that contains in amino acid or the dna sequence dna with the minimum difference of stipulating in the drawings intended being included in this term.This species diversity can be caused by the mutagenesis incident of not obvious interferencing protein linkage function.
[030] in one embodiment of the present invention, under following condition, carry out strict hybridization: a) hybridization: the calf thymus DNA that 0.75M NaCl, 0.15Tris HCl, 10mM EDTA, 0.1%NaCl, 0.1%SLS, 0.03%BSA, 0.03%Ficoil 400,0.03%PVP and 100 μ g/ml boil, 50 ℃ down about 12 hours and; B) with 0.1X SET, 0.1%SDS, 0.1%NaCl and 0.1M phosphate buffered saline buffer, washed 3 times totally 30 minutes down, and go up the existence of the DNA that detects double-stranded hybridization at southern blotting technique (Southern Blot) at 45 ℃.
[031] U.S. Provisional Patent Application of submitting in all reference of quoting of this paper and on September 15th, 2005 is introduced into as a reference for the 60/717th, No. 296.
Embodiment
Example I: use degenerated primer to clone certain (strain 9 deg n-7) dna ligase gene of thermophilic Coccus
[032] at first by the genomic dna amplification gene of PCR from thermophilic Coccus certain (strain 9 deg n-7).Obtain the forward primer sequence from the reference of FEMS Microbiology Lett.236 (2): 267-273 (2004) such as J.Bact.182 such as Nakatani (22): 6424-6433 (2000) (the thermophilic coccus of Ke Dakala) and Rolland (the thermophilic coccus of happiness cigarette).It is as follows to have the consensus sequence design of specifying degeneracy:
Forward primer
5′CGGTGGTGCATATGRGCGAYATGMRSTACTC(SEQ?ID?NO：20)
Reverse primer
5′ATAAACTCTAGATTACYTCTTCGCCTTGAACCTCTCCTGG(SEQ?ID?NO：21)
[033] uses the gene of the primer of thermophilic Coccus certain (strain 9 deg n-7) from the genomic dna DNA amplification ligase enzyme of thermophilic Coccus certain (strain 9 deg n-7).It is as follows to be used for the PCR reaction conditions of clone gene:
[034] 100 μ l reaction mixture---contain 20mM Tris-HCL, 10mM KCl, the 10mM (NH of pH 8.8
4)
2SO
4With 4mM MgSO
4, the TaqDNA polysaccharase of genomic dna, forward and every kind of 500ng of reverse primer, 2.5 units of the thermophilic Coccus of every kind of dNTP, 50ng certain (strain 9 deg n-7) of 0.1%Triton X-100,200 μ M and 0.02 unit
Archaeal dna polymerase, be heated to 94 ℃ 1 minute, then 45 ℃ 1 minute, and then 72 ℃ 3 minutes.Repeat 30 times this temperature cycle.After finishing circulation, temperature of reaction is reduced to room temperature, and adds the 5 e. coli dna polymerase Klenow of unit fragments, and further incubation 5 minutes at room temperature.Then, reactant is adjusted into 70mM EDTA.The PCR product is with phenol extraction, with alcohol precipitation, and goes up its desalination at CL6B agarose spin post (sepharose spincolumn).
[035] with 1700bp PCR product cloning in intestinal bacteria.The litmus 28i of EcoRV-cutting is used as the segmental carrier of cloned DNA.
[036] 10 μ l ligation things in the T4DNA ligase enzyme damping fluid comprise 80ng insert fragment, 80ng litmus carrier and 400 T4DNA of unit ligase enzymes (New England Biolabs, Inc., Ipswich, MA).Ligation is incubated overnight at 16 ℃, enters intestinal bacteria TB1 cell through electroporation, and on IPTG XGAL flat board bed board.
[037] selects white bacterium colony.One in 9 white colonies has 1700bp and inserts fragment.Independently electroporation produces and has segmental another clone of 1700bp insertion.Insertion fragment among these two clones is checked order.
[038] from these clones' sequence, the new forward primer design with less degeneracy is as follows:
9 ° of N forward primers:
5′cggtggtgcatatgggcgayatgaggtactccgagctgg(SEQ?ID?NO：22)
(2) second forward primer that uses the primer of ratio in embodiment (1) to have less degeneracy is cloned the ligase enzyme of thermophilic Coccus certain (strain 9 deg n-7)
[039] use 9 ° of N-7 forward primers to implement other 4 independently PCR reactions, its only comprise a degeneracy base replaced in above-mentioned (1) forward primer---it contains 5 degeneracy bases.
[040] 100 μ l contains every kind of dNTP and 1 μ l Phusion archaeal dna polymerase (the New England Biolabs of certain (strain 9 deg n-7) genomic dna of the thermophilic Coccus of 50ng, forward and every kind of 500ng of reverse primer, 200 μ M, Inc, Ipswich, MA) Phusion HF damping fluid (New England Biolabs, Inc, Ipswich, MA) be heated to 98 ℃ and continue 30 seconds, do the circulation of 25 following processes then: 98 ℃ 10 seconds, then 70 ℃ 30 seconds, next 72 ℃ 1 minute.Then 72 ℃ of incubation reaction things 5 minutes.The product of each PCR reaction is handled as initial p CR reactant, and is cloned among the litmus 28i, as mentioned above.By preparing DNA in a small amount, two independent clonings (A1 and A3) that obtain from PCR reaction are proved and contain 1700 base pairs and insert fragment, and are proved equally from each clones who obtains of other three PCR reactions (B2, C3, D3) and contain 1700 base pairs and insert fragment.Cultivate these clones then, and with their crude extract electrophoresis on SDS PAGE.The protein of each clonal expression 60kd.
[041] be purified from clone A1, A3, B2, C3, plasmid that D3 obtains and other lig7 and lig8, and to inserting sequencing fragment.In 1a-5, provide dna sequence dna (SEQ ID NOS:1-7) at Fig. 1 a-1.
[042] although without wanting to be limited by theory, observed little difference can be explained by the clonal vaviation in certain (strain 9 deg n-7) cell mass of thermophilic Coccus in sequence.Sequence variations is all the 3rd change in location, and perhaps conserved amino acid changes.Clone B2 is the representative of ligase enzyme consensus sequence.Dna ligase is expressed (Fig. 2) first in closely-controlled expression vector.
(3) at expression in escherichia coli ligase enzyme gene (B2)
[043] by with NdeI and XbaI cutting, shears the B2 fragment from the litmus carrier.Downcut the 1700bp fragment from sepharose, and digest gel slice to discharge described fragment with gelase.
[044] (Ipswich MA), and makes its dephosphorylation for NewEngland Biolabs, Inc. by preparing expression vector pMalC2X with NdeI and XbaI cutting.Contain in the reactant that 400ng inserts carrier in fragment and the 100ng T4DNA ligase enzyme damping fluid and 200 T4DNA of unit ligase enzymes at 10 μ l,, the 1700bp base pair PCR fragment of cutting is connected to the pMalC2X carrier 16 ℃ of incubations 16 hours.Ligation thing electroporation is gone into intestinal bacteria TB1 cell, and carry separated and certain (strain 9 deg n-7) B2-1 (Fig. 3) of the thermophilic Coccus of called after of the segmental clone of 1700bp.
[045] growth is cloned in the LB substratum, and induces with IPTG.The sample of inductive cell is cleaved, and on SDS PAGE gel electrophoresis, be with accordingly with the protein that demonstrates with about 60kd size.The protein that the analysis revealed genes encoding of the protein sequence that obtains from dna sequence dna is had 26 seldom used arginine codons.Therefore, (Stratagene, LaJoIIa CA) are used to obtain higher levels of expression to contain the host cell (intestinal bacteria BL-2 (DE3) RIL) that is useful on arginic seldom used tRNA.After thermophilic Coccus certain (strain 9 deg n-7) was induced, the B2-1 plasmid in host's sample was analyzed by SDS PAGE, and observed tangible 60kd band.
(4) comparison of certain (strain 9 deg n-7) ligase enzyme of thermophilic Coccus and other heat-stable DNA ligase enzyme
[046] by the comparison of CLUSTAL multisequencing, certain (strain 9 deg n-7) dna ligase aminoacid sequence of thermophilic Coccus and other 4 kinds of thermophilic dna ligases are compared.CLUSTAL W (1.82) multisequencing comparison (Multiple Sequence Alignments) sequence form is Pearson.
Sequence 1:9 ° of N-7-B2 (SEQ ID NO:15) 564aa
The thermophilic coccus of sequence 2: Ke Dakala (SEQ ID NO:16) 562aa
Sequence 3: deep-sea hot-bulb bacterium (SEQ ID NO:17) 559aa
Sequence 4: fierce hot-bulb bacterium (SEQ ID NO:18) 561aa
Sequence 5: the happiness thermophilic coccus of cigarette (SEQ ID NO:19) 559aa
The identity score
Sequence (1: 2) comparison score: 90
Sequence (1: 3) comparison score: 81
Sequence (1: 4) comparison score: 78
Sequence (1: 5) comparison score: 88
Sequence (2: 3) comparison score: 80
Sequence (2: 4) comparison score: 80
Sequence (2: 5) comparison score: 87
Sequence (3: 4) comparison score: 90
Sequence (3: 5) comparison score: 78
Sequence (4: 5) comparison score: 77
[048] provides comparison among Fig. 4.The known immediate relatives of certain (strain 9 deg n-7) dna ligase of thermophilic Coccus are the thermophilic coccus dna ligases of Ke Dakala, wherein have 90% amino acid identity and 80.9% Nucleotide identity.
(5) purifying of certain (strain 9 deg n-7) dna ligase of thermophilic Coccus
[049] uses the segmental pMalC2X plasmid of B2 (the New England Biolabs contain the dna ligase that obtains from thermophilic Coccus certain (strain 9 deg n-7), Inc., Ipswich, MA) transformed into escherichia coli BL-21 (DE3)-RIL (Stratagene, La Jolla, CA).In the 100ml LB substratum of the paraxin of penbritin that contains 50 μ g/ml and 25 μ g/ml, under 37 ℃, grown cell.After being incubated overnight, culture being transferred in 10 liters of fermentor tanks, and,, and added 0.1 IPTG that restrains up to the OD600 that reaches 0.59 at 37 ℃ of following incubations.Other 5.75 hours of incubation culture, and results.At-20 ℃, preserve this cell mass (cell paste).
[050] 10 gram cell masses in the 10mM of 40ml pH7.5 Tris HCl, 20mM NaCl, 0.1mMEDTA and 1.0mM DTT are thawed and are passed through ultrasonic treatment.Extract is inserted 0.3mM PMSF and 200mM NaCl.By the centrifugal purification extract.With the extract that purifies by the DEAE agarose column among the 0.2M NaCl.Collect the protein that flows through this post then, and its dilution is the NaCl of 100mM.It is administered to phosphocellurose column, and arrives the absorbed protein of NaCl gradient elution of 1.1M with 100mM.Analyze this fraction (Fig. 5) by SDS PAGE, collect the main peak of 60kd, and be heated 75 ℃, 30 minutes.By this solution of centrifugal purification, be 100mMNaCl with the solution dilution that purifies, and it is administered to hydroxyapatite column.The ammonium sulfate of 0-13% gradient is applied to this post, collect fraction, and by at T4DNA ligase enzyme damping fluid (the New England Biolabs of the HindIII λ DNA that contains 50 μ g/ml as substrate, Inc., Ipswich, MA) in a plurality of fractions of incubation, analyze the activity of these fractions.Under 37 ℃, this reactant of incubation 10 minutes.By adding 10% 100mM EDTA and 50% glycerine and bromjophenol blue dyestuff termination reaction.Anti-primer is heated to 65 ℃, and it is loaded on 1% the sepharose analyzes.
[051] collect and to contain the active pipe of about 80% ligase enzyme, and with it at the 10mM of 50% glycerine, pH7.5 Tris HCl, 50mM KCl, 10mM (NH
4)
2SO
4, dialyse among 0.1mM EDTA and the 1.0mM DTT.Certain (strain 9 deg n-7) dna ligase of the thermophilic Coccus of purifying is stored in-20 ℃.
Embodiment 2: the characteristic of certain (strain 9 deg n-7) dna ligase of thermophilic Coccus
[052] reaction conditions of Tui Jianing is:
10mM?Tris-HCl，pH7.5
2.5mM?MgCl
2
2.5mM?DTT
300μMATP
[053] under 45 ℃, being used to analyze active typical substrate is λ DNA.Suitably the λ DNA of digestion can illustrate the connection state that the 12-base is extended at λ DNA end.Usually, we use HindIII or the predigested λ DNA of BstEII.On agarose gel electrophoresis, implement to connect to analyze.
[054] Km of ATP is presented at below the 100 μ M.Utilize Triton X-100 to stimulate this activity.
[055] different with the thermophilic coccus dna ligase of happiness cigarette, there is NAD in certain (strain 9 deg n-7) ligase enzyme of thermophilic Coccus
+Situation under, do not have detectable activity.
[056] this enzyme require magnesium ion.2.5mM MgCl
2Acquisition is than 10mM MgCl
2Many 10 times activity.
[057] after 30 minutes, keeps the activity (Fig. 6) between 25% to 50% at about this enzyme of 100 ℃ of following incubations.
[058] under 90 ℃, ligase enzyme can seal nicked DNA.Dna ligase is with the nicked pUC19 plasmid DNA of BstNBI incubation, and the band otch plasmid that will relax changes into covalently closed circular DNA, as determining by agarose gel electrophoresis.Speed of reaction height when the speed of reaction ratio is at 45 ℃ in the time of 80 ℃.Although nicked plasmid in the sex change of 90 ℃ of experience, at 90 ℃ a large amount of otch sealings takes place before sex change, this changes all nicked plasmids into single stranded plasmid.
Embodiment 3: the application of certain (strain 9 deg n-7) dna ligase of thermophilic Coccus in DNA reparation mixture
[059] use comprises the mixture of the enzyme of strain 9 deg n-7DNA ligase enzyme, repairs the depurination destructive DNA of institute.
[060] DNA is by Ide in experiment reaction, and Biochemistry such as H 32 (32): the depurination that 8276-83 (1993) describes and impaired.λ DNA (NEB#N3011, New EnglandBiolabs, Inc., Ipswich is MA) by the alcohol precipitation.DNA is resuspended in the depurination damping fluid (100mM NaCl, 10mM Citrate trianion, pH 5.0) with the concentration of 0.5mg/ml, and 70 ℃ of following incubations 120 minutes.Then sample alcohol is precipitated, and be suspended in 0.01MTris, 0.001M EDTA again, in the solution of pH 8.0.After with damping fluid contrast calibration, contain the A of dna solution by measurement
260Determine the concentration of DNA.
[061] in the mixture of following enzyme, at room temperature the impaired DNA of incubation reaches 10 minutes: DNA (1ng); 100 μ M dNTPs (NEB#M0447, New England Biolabs, Ipswich, MA); 1mM ATP; 480 Taq of unit ligase enzymes (NEB#M0208, New EnglandBiolabs, Ipswich, MA) or 9 ° of N-7DNA ligase enzymes of 500 units (NEB#M0238, New England Biolabs, Ipswich, MA); 0.1 the e. coli dna polymerase I of unit (intestinal bacteria polI) (NEB#M0209, New England Biolabs, Inc., Ipswich, MA); The 10 intestinal bacteria restriction endonuclease IV of unit (NEB#M0304, New England Biolabs, Inc., Ipswich, MA); 1 * Thermopol damping fluid (NEB#B9004, New England Biolabs, Inc., Ipswich, MA), to final volume 47.5 μ L.
[062], sample transfer on ice, is increased then in the reaction ending.Negative control is as above handled, but does not use enzyme.
Dna amplification reaction
[063] according to the method for Nucl.Acids Res.32:1197-1207 (2004) such as Wang, use following primer, implement the amplification of λ DNA: CGAACGTCGCGCAGAGAAACAGG (L72-5R) (SEQ ID NO:23) and CCTGCTCTGCCGCTTCACGC (L30350F) (SEQ ID NO:24).
[064] amplification mixture of 2.5 μ l is joined in 47.5 the above-mentioned reparation mixture.This amplification mixture contain 100 μ M dNTPs, 5 Taq of unit archaeal dna polymerases (New EnglandBiolabs, Inc., Ipswich, MA), 0.1 Vent of unit
(exo+) archaeal dna polymerase (NewEngland Biolabs, Inc., Ipswich, MA), 5 * 10
-11M primer L72-5R and 5 * 10
-11M primer L30350F is in 1 * Thermopol damping fluid.
[065], when repair enzyme omits from reaction, the store buffer liquid of proper volume is joined in the reactant in order to correct the influence of enzyme store buffer liquid.In all cases, use following parameters in thermal cycler (thermal cycler), to carry out amplified reaction: 95 ℃ of 1 circulations in following 20 seconds, next 94 ℃ following 5 seconds, then 72 ℃ of 25 circulations in 5 minutes.The size of the amplicon that is amplified is 5kb.
[066], determines the result of DNA (5kb) amplification by 1% agarose gel electrophoresis.With 6 * carried dye (Molecular Cloning:A Laboratory Manual, 3rd ed., eds.Sambrook and Russell, Cold Spring Harbor Press, Cold Spring Harbor, NY, 2001, pp.5.4-5.17) join in the 50 μ l amplified reaction things.Then, with 20 these solution of μ l and as the 2-logarithm gradient molecular weight standard (2-logladder) of 1 μ g of dimensional standard (NEB#N3200, New England Biolabs, Inc., Ipswich MA) is loaded on the sepharose together.
Sequence table
＜110〉New England Biolabs, Inc. (US) Massachusetts, United States of America
I. Anthony Heald Ke Laote
E. Anthony Heald Ke Laote
＜120〉discovery, clone and the purifying of certain (strain 9 deg n-7) dna ligase of thermophilic Coccus
<130>NEB-268-PCN
<150>60/717,296
<151>2005-09-15
<150>PCT/US06/35919
<151>2006-09-15
<160>24
<170>PatentIn?version?3.4
<210>1
<211>1695
<212>DNA
＜213〉the unknown
<220>
＜223〉certain (Thermococcus sp.) strain 9 deg n of thermophilic Coccus
<400>1
atgggcgata?tgaggtactc?cgagctggcc?gaactctacc?ggaggcttga?gaagaccacg?60
ctcaaaacgc?tcaagaccaa?gttcgtcgcg?gacttcctca?agaaaacacc?cgacgatttg?120
ctcgagatag?ttccctacct?gattctcggc?aaggtctttc?cggactggga?cgagcgcgag?180
ctcggcgtcg?gcgagaagct?tctcataagg?gccgtttcga?tggcaaccgg?cgtccccgag?240
agggaaatcg?agaactcgat?taaggacacc?ggcgatttgg?gcgagagcgt?ggctctggct?300
ctaaagaaga?ggaagcagaa?gagcttcttc?agccagcccc?tcacgataaa?gcgcgtctac?360
agcaccttcg?ttaaggttgc?cgaggcgagc?ggagagggga?gccaggacag?gaagatgaag?420
tacttagcaa?acctcttcat?ggatgctcaa?cccgaggagg?gcaagtacat?agccagaacc?480
gtcctcggga?cgatgaggac?gggcgtcgcc?gagggaatcc?tgcgcgatgc?catagcggag?540
tccttcaagg?tgaagccaga?gctcgtcgag?agggcctaca?tgctcacgag?cgacttcggc?600
tacgtggcga?aggtcgccaa?gctcgagggg?aacgaggggc?tctcgaaggt?cagcatacag?660
attgggaagc?cgataaggcc?gatgctagct?caaaacgccg?ccaacgtcaa?ggaagcgcta?720
atcgagatgg?gcggtgaggc?ggccttcgag?attaagtacg?acggcgcgcg?cgttcaggtc?780
caccgcgatg?gggacagggt?gataatctac?tcgaggaggc?ttgagaacgt?cacccgctcg?840
attcctgaga?tagttgaggc?ggtaaaggcc?tccctgaagc?cctctaatgt?catagtcgag?900
ggcgagctgg?ttgccgtcgg?cgagaacggt?cgcccgaggc?ccttccagta?cgtcctgagg?960
cgctttagga?ggaagtacaa?catcgaggag?atgattgaga?agattccgct?cgagctcaac?1020
ctcttcgaca?tcctctacgt?tgaaggagag?agcctcatcg?acacgaagtt?cgcagagagg?1080
aggaagaagc?tcgaggagag?cgttgaggag?agcgataaga?taaagctcgc?cgaacagctc?1140
gttacgaaga?aggttgaaga?ggccgaggag?ttctacaaga?gggcccttga?gctcggccac?1200
gagggcctga?tggcgaagag?gctggactcc?atctacgagc?ccggaaaccg?cggtaagaag?1260
tggctgaaga?ttaagcccac?gatggagaac?cttgacctcg?tcattatcgg?agccgaatgg?1320
ggcgagggca?ggcgcgcgca?cctgctcggc?tcgttcctcg?ttgcggccta?cgaccccgag?1380
agcggtgagt?tcgtcccggt?gggcaaggtc?gggagcggtt?tcaccgatga?agatttggtc?1440
gagttcacca?agatgctcaa?gcccctgatt?gtccgtgaag?agggcaagtt?cgtcgagatt?1500
gagcccaagg?tcgtcatcga?ggtcacctac?caggagatac?agaagagccc?caagtataag?1560
agcggtttcg?cgcttcgctt?cccgcgctac?gtggcgctga?gggaagataa?aagcccggag?1620
gaggctgaca?ccatagagag?ggtcgcccag?ctctacgagc?tccaggagag?gttcaaggcg?1680
aagaggtaat?ctaga 1695
<210>2
<211>1695
<212>DNA
＜213〉the unknown
<220>
＜223〉certain strain 9 deg n of thermophilic Coccus
<400>2
atgggcgata?tgaggtactc?cgagctggcc?gaactctacc?ggaggcttga?gaagaccacg?60
ctcaaaacgc?tcaagaccaa?gttcgtcgcg?gacttcctca?agaaaacacc?cgacgatttg?120
ctcgagatag?ttccctacct?gattctcggc?aaggtctttc?cggactggga?cgagcgcgag?180
ctcggcgtcg?gcgagaagct?tctcataagg?gccgtttcga?tggcaaccgg?cgtccccgag?240
agggaaatcg?agaactcgat?taaggacacc?ggcgatttgg?gcgagagcgt?ggctctggct?300
ctaaagaaga?ggaagcagaa?gagcttcttc?agccagcccc?tcacgataaa?gcgcgtctac?360
agcaccttcg?ttaaggttgc?cgaggcgagc?ggagagggga?gccaggacag?gaagatgaag?420
tacttagcaa?acctcttcat?ggatgctcaa?cccgaggagg?gcaagtacat?agccagaacc?480
gtcctcggga?cgatgaggac?gggcgtcgcc?gagggaatcc?tgcgcgatgc?catagcggag?540
tccttcaagg?tgaagccaga?gctcgtcgag?agggcctaca?tgctcacgag?cgacttcggc?600
tacgtggcga?aggtcgccaa?gctcgagggg?aacgaggggc?tctcgaaggt?cagcatacag?660
attgggaagc?cgataaggcc?gatgctagct?caaaacgccg?ccaacgtcaa?ggaagcgcta?720
atcgagatgg?gcggtgaggc?ggccttcgag?attaagtacg?acggcgcgcg?cgttcaggtc?780
caccgcgatg?gggacagggt?gataatctac?tcgaggaggc?ttgagaacgt?cacccgctcg?840
attcctgaga?tagttgaggc?ggtaaaggcc?tccctgaagc?cctctaatgt?catagtcgag?900
ggcgagctgg?ttgccgtcgg?cgagaacggt?cgcccgaggc?ccttccagta?cgtcctgagg?960
cgctttagga?ggaagtacaa?catcgaggag?atgattgaga?agattccgct?cgagctcaac?1020
ctcttcgaca?tcctctacgt?tgaaggagag?agcctcatcg?acacgaagtt?cgcagagagg?1080
aggaagaagc?tcgaggagag?cgttgaggag?agcgataaga?taaagctcgc?cgaacagctc?1140
gttacgaaga?aggttgaaga?ggccgaggag?ttctacaaga?gggcccttga?gctcggccac?1200
gagggcctga?tggcgaagag?gctggactcc?atctacgagc?ccggaaaccg?cggtaagaag?1260
tggctgaaga?ttaagcccac?gatggagaac?cttgacctcg?tcattatcgg?agccgaatgg?1320
ggcgagggca?ggcgcgcgca?cctgctcggc?tcgttcctcg?ttgcggccta?cgaccccgag?1380
agcggtgagt?tcgtcccggt?gggcaaggtc?gggagcggtt?tcaccgatga?agatttggtc?1440
gagttcacca?agatgctcaa?gcccctgatt?gtccgtgaag?agggcaagtt?cgtcgagatt?1500
gagcccaagg?tcgtcatcga?ggtcacctac?caggagatac?agaagagccc?caagtataag?1560
agcggtttcg?cgcttcgctt?cccgcgctac?gtggcgctga?gggaagataa?aagcccggag?1620
gaggctgaca?ccatagagag?ggtcgcccag?ctctacgagc?tccaggagag?gttcaaggcg?1680
aagaagtaat?ctaga 1695
<210>3
<211>1695
<212>DNA
＜213〉the unknown
<220>
＜223〉certain strain 9 deg n of thermophilic Coccus
<400>3
atgggcgata?tgaggtactc?cgagctggcc?gaactttaca?gaaggcttga?gaagaccacg?60
ctcaaaacgc?tcaagaccaa?gtttgtcgcg?gacttcctca?agaaaactcc?cgacgatttg?120
ctcgagatag?ttccctacct?gattctcggc?aaggtctttc?cggactggga?cgagcgcgag?180
ctcggcgtcg?gggaaaagtt?gctcataagg?gccgtttcga?tggcaaccgg?cgtccccgag?240
agggaaatcg?agaactcgat?taaggacacg?ggcgatttgg?gcgagagcgt?ggctctggct?300
ctaaagaaga?ggaagcagaa?gagcttcttc?agccagcccc?tcacgataaa?gcgcgtctac?360
agcaccttcg?ttaaggttgc?cgaggcgagc?ggagagggga?gccaggwcag?gaagatgaag?420
tacttagcaa?atctcttcat?ggatgctcaa?cccgaggagg?gcaagtacat?agccagaacc?480
gtcctcggaa?cgatgaggac?gggcgtcgcc?gagggaatcc?tgcgcgatgc?catagcggag?540
gccttcaagg?tgaagccaga?gctcgtcgag?agggcctaca?tgctcacgag?cgacttcggc?600
tacgtggcga?aggtcgccaa?gctcgagggg?aacgaggggc?tctcgaaggt?cagcatacag?660
attgggaagc?cgataaggcc?gatgctagct?caaaacgccg?ccaacgtcaa?ggaagcgcta?720
atcgagatgg?gcggtgaggc?ggccttcgag?attaagtacg?acggcgcgcg?cgttcaggtc?780
caccgcgatg?gggacagggt?gataatctac?tcgaggaggc?ttgagaacgt?cacccgctcg?840
attcctgaga?tagttgaggc?ggtaaaggcc?tccctgaagc?cctctaatgt?catagtcgag?900
ggcgagctgg?ttgccgtcgg?cgagaacggt?cgcccgaggc?ccttccagta?cgtcctgagg?960
cgctttagga?ggaagtacaa?catcgaggag?atgattgaga?agattccgct?cgagctcaac?1020
ctcttcgaca?tcctctacgt?tgaaggggag?agcctcattg?acacgaagtt?cgccgagagg?1080
aggaggaagc?ttgaggagag?cgtcgaggag?ggcgataaga?taaagctcgc?cgaacagctc?1140
gttacgaaga?aggtcgaaga?ggccgaggag?ttctacaaga?gggcccttga?gctcggccac?1200
gagggcctga?tggcgaagag?gctggactcc?atctacgagc?ccggaaaccg?cggtaagaag?1260
tggctgaaga?ttaagcccac?gatggagaac?cttgacctcg?tcattatcgg?agccgaatgg?1320
ggcgagggga?ggcgcgcgca?cctgcttggc?tcgttcctcg?ttgcggccta?cgaccccgag?1380
agcggtgagt?tcgtcccggt?gggcaaggtc?gggagcggtt?tcaccgatga?agatttggtc?1440
gagttcacca?agatgctcaa?gcccctgatt?gtccgtgaag?agggcaagtt?cgtcgagatt?1500
gagcccaagg?tcgtcatcga?ggtcacctac?caggagatac?agaagagccc?caagtataag?1560
agcggtttcg?cgcttcgctt?cccgcgctac?gtggcgctga?gggaagataa?aagcccggag?1620
gaggctgaca?ccatagagag?ggtcgcccag?ctctacgagc?tccaggagag?gttcaaggcg?1680
aagaagtaat?ctaga 1695
<210>4
<211>1695
<212>DNA
＜213〉the unknown
<220>
＜223〉certain strain 9 deg n of thermophilic Coccus
<400>4
atgggcgata?tgaggtactc?cgagctggcc?gaactctacc?ggaggcttga?gaagaccacg?60
ctcaaaacgc?tcaagaccaa?gttcgtcgcg?gacttcctca?agaaaacacc?cgacgatttg?120
ctcgagatag?ttccctacct?gattctcggc?aaggtctttc?cggactggga?cgagcgcgag?180
ctcggcgtcg?gggaaaagtt?gctcataagg?gccgtttcga?tggcgactgg?ggttccagag?240
agggaaatcg?agaactcgat?taaggacacg?ggcgatttgg?gcgagagcgt?ggccttggcc?300
ctaaagaaga?ggaagcagaa?gagcttcttc?agccagcccc?tcacgataaa?gcgcgtctac?360
agcaccttcg?ttaaggttgc?cgaggcgagc?ggagagggga?gccaggacag?gaagatgaag?420
tacttagcaa?acctcttcat?ggatgctcaa?cccgaggagg?gcaagtacat?agccagaacc?480
gtcctcggga?cgatgaggac?gggcgtcgcc?gagggaatcc?tgcgcgatgc?catagcggag?540
gccttcaagg?tgaagccaga?gctcgtcgag?agggcctaca?tgctcacgag?cgacttcggc?600
tacgtggcga?aggttgcaaa?gctcgagggg?aacgaggggc?tctcaaaggt?tagcatacag?660
attgggaagc?cgataaggcc?gatgctagct?caaaacgccg?ccaacgtcaa?ggaagcgcta?720
atcgagatgg?gcggtgaggc?ggccttcgag?attaagtacg?acggcgcgcg?cgttcaggtc?780
caccgcgatg?gggacagggt?gataatctac?tcgaggaggc?ttgagaacgt?cacccgctcg?840
attcctgaga?tagttgaggc?ggtaaaggcc?tccctgaagc?cctctaatgt?catagtcgag?900
ggcgagctgg?ttgccgtcgg?cgagaacggt?cgcccgaggc?ccttccagta?cgtcctgagg?960
cgctttagga?ggaagtacaa?catcgaggag?atgattgaga?agattccgct?cgagctcaac?1020
ctcttcgaca?tcctctacgt?tgaaggagag?agcctcatcg?acacgaagtt?cgcagagagg?1080
aggaagaagc?tcgaggagag?cgttgaggag?agcgataaga?taaagctcgc?cgaacagctc?1140
gttacgaaga?aggtcgaaga?ggccgaggag?ttctacaaga?gggcccttga?gctcggccac?1200
gagggcctga?tggcgaagag?gctggactcc?atctacgagc?ccggaaaccg?cggtaagaag?1260
tggctgaaga?ttaagcccac?gatggagaac?cttgacctcg?tcattatcgg?agccgaatgg?1320
ggcgagggga?ggcgcgcgca?cctgctcggc?tcgttcctcg?ttgcggccta?cgaccccgag?1380
agcggtgagt?tcgtcccggt?gggcaaggtc?gggagcggtt?tcaccgatga?agatttggtc?1440
gagttcacca?agatgctcaa?gcccctgatt?gtccgtgaag?agggcaagtt?tgtcgagatt?1500
gagcccaagg?tcgtcatcga?ggtcacctac?caggagatac?agaagagccc?caagtataag?1560
agcggtttcg?cgcttcgctt?cccgcgctac?gtggcgctga?gggaagataa?aagcccggag?1620
gaggccgaca?ccatagagag?ggtcgcccag?ctctacgagc?tccaggagag?gttcaaggcg?1680
aagaagtaat?ctaga 1695
<210>5
<211>1695
<212>DNA
＜213〉the unknown
<220>
＜223〉certain strain 9 deg n of thermophilic Coccus
<400>5
atgggcgata?tgaggtactc?cgagctggcc?gaactctacc?ggaggcttga?gaagaccacg?60
ctcaaaacgc?tcaagaccaa?gttcgtcgcg?gacttcctca?agaaaacacc?cgacgatttg?120
ctcgagatag?ttccctacct?gattctcggc?aaggtctttc?cggactggga?cgagcgcgag?180
ctcggcgtcg?gggaaaagtt?gctcataagg?gccgtttcga?tggcgactgg?ggttccagag?240
agggaaatcg?agaactcgat?taaggacacg?ggcgatttgg?gcgagagcgt?ggccttggcc?300
ctaaagaaga?ggaagcagaa?gagcttcttc?agccagcccc?tcacgataaa?gcgcgtctac?360
agcaccttcg?ttaaggttgc?cgaggcgagc?ggagagggga?gccaggacag?gaagatgaag?420
tacttagcaa?acctcttcat?ggatgctcaa?cccgaggagg?gcaagtacat?agccagaacc?480
gtcctcggga?cgatgaggac?gggcgtcgcc?gagggaatcc?tgcgcgatgc?catagcggag?540
gccttcaagg?tgaagccaga?gctcgtcgag?agggcctaca?tgctcacgag?cgacttcggc?600
tacgtggcga?aggttgcaaa?gctcgagggg?aacgaggggc?tctcaaaggt?tagcatacag?660
attgggaagc?cgataaggcc?gatgctagct?caaaacgccg?ccaacgtcaa?ggaagcgcta?720
atcgagatgg?gcggtgaggc?ggccttcgag?attaagtacg?acggcgcgcg?cgttcaggtc?780
caccgcgacg?gggatagggt?gataatatac?tcgaggaggc?ttgagaacgt?cacccgctcg?840
attcctgaga?tagtcgaggc?ggtaaaggcc?tccctgaagc?cttctaaggt?catagtcgag?900
ggcgagctgg?ttgccgtcgg?cgagaacggt?cgcccgaggc?ccttccagta?cgtcctcagg?960
aggttcagga?ggaagtacaa?catcgaggag?atgattgaga?agattccgct?cgagctcaac?1020
ctcttcgata?tcctctacgt?tgatggggag?agtctcatcg?acacgaagtt?cgcagagagg?1080
aggaagaaac?tcgaggagag?tgttgaggag?agcgataaga?taaagctcgc?cgaacagctc?1140
gttacgaaga?aggtcgaaga?ggccgaggag?ttctacaaga?gggcccttga?gctcggccac?1200
gagggcctga?tggcgaagag?gctggactcc?atctacgagc?ccggaaaccg?cggtaagaag?1260
tggctgaaga?ttaagcccac?gatggagaac?cttgacctcg?tcattatcgg?agccgaatgg?1320
ggcgagggga?ggcgcgcgca?cctgcttggc?tcgttcctcg?ttgcggccta?cgaccccgag?1380
agcggtgagt?tcgtcccggt?gggcaaggtc?gggagcggtt?tcaccgatga?agatttggtc?1440
gagttcacca?agatgctcaa?gcccctgatt?gtccgtgaag?agggcaagtt?tgtcgagatt?1500
gagcccaagg?tcgtcatcga?agtaacgtat?caggagatac?agaagagccc?caagtataag?1560
agcggtttcg?cgcttcgctt?cccgcgctac?gtggcgctga?gggaagataa?aagcccggag?1620
gaggctgaca?ccatagagag?ggtcgcccag?ctctacgagc?tccaggagag?gttcaaggcg?1680
aagaagtaat?ctaga 1695
<210>6
<211>1695
<212>DNA
＜213〉the unknown
<220>
＜223〉certain strain 9 deg n of thermophilic Coccus
<400>6
atgggcgaca?tgaggtactc?cgagctggcc?gaactctacc?ggaggcttga?gaagaccacg 60
ctcaaaacgc?tcaagaccaa?gttcgtcgcg?gacttcctca?agaaaacacc?cgacgatttg?120
ctcgagatag?ttccctacct?gattctcggc?aaggtctttc?cggactggga?cgagcgcgag?180
ctcggcgtcg?gggaaaagtt?gctcataagg?gccgtttcga?tggcgactgg?ggttccagag?240
agggaaatcg?agaactcgat?taaggacacg?ggcgatttgg?gcgagagcgt?ggccttggcc?300
ctaaagaaga?ggaagcagaa?gagcttcttc?agccagcccc?tcacgataaa?gcgcgtctac?360
agcaccttcg?ttaaggttgc?cgaggcgagc?ggagagggga?gccaggacag?gaagatgaag?420
tacttagcaa?acctcttcat?ggatgctcaa?cccgaggagg?gcaagtacat?agccagaacc?480
gtcctcggga?cgatgaggac?gggcgtcgcc?gagggaatcc?tgcgcgatgc?catagcggag?540
gccttcaagg?tgaagccaga?gctcgtcgag?agggcctaca?tgctcacgag?cgacttcggc?600
tacgtggcga?aggttgcaaa?gctcgagggg?aacgaggggc?tctcaaaggt?tagcatacag?660
attgggaagc?cgataaggcc?gatgctagct?caaaacgccg?ccaacgtcaa?ggaagcgcta?720
atcgagatgg?gcggtgaggc?ggccttcgag?attaagtacg?acggcgcacg?cgttcaggtc?780
caccgcgacg?gggacagggt?gataatctac?tcgaggaggc?ttgagaacat?cacccgctcg?840
attcctgaga?tagtcgaggc?ggtaaaggcc?tccctgaagc?cttctaaggt?catagtcgag?900
ggcgagctgg?ttgccgtcgg?cgagaacggt?cgcccgaggc?ccttccagta?cgtcctcagg?960
aggttcagga?ggaagtacaa?catcgaggag?atgattgaga?agattccgct?cgagctcaac?1020
ctcttcgata?tcctctacgt?tgatggggag?agtctcatcg?acacgaagtt?cgcagagagg?1080
aggaagaaac?tcgaggagag?tgttgaggag?agcgataaga?taaagctcgc?cgaacagctc?1140
gttacgaaga?aggtcgaaga?ggccgaggag?ttctacaaga?gggcccttga?gctcggccac?1200
gagggcctga?tggcgaagag?gctggactcc?atctacgagc?ccggaaaccg?cggtaagaag?1260
tggctgaaga?ttaagcccac?gatggagaac?cttgacctcg?tcattatcgg?agccgaatgg?1320
ggcgagggga?ggcgcgcgca?cctgcttggc?tcgttcctcg?ttgcggccta?cgaccccgag?1380
agcggtgagt?tcgtcccggt?gggcaaggtc?gggagcggtt?tcaccgatga?agatttggtc?1440
gagttcacca?agatgctcaa?gcccctgatt?gtccgtgaag?agggcaagtt?tgtcgagatt?1500
gagcccaagg?tcgtcatcga?agtaacgtat?caggagatac?agaagagccc?caagtataag?1560
agcggtttcg?cgcttcgctt?cccgcgctac?gtggcgctga?gggaagataa?aagcccggag?1620
gaggctgaca?ccatagagag?ggtcgcccag?ctctacgagc?tccaggagag?gtacaaggcg?1680
aagaggtaat?ctaga 1695
<210>7
<211>1695
<212>DNA
＜213〉the unknown
<220>
＜223〉certain strain 9 deg n of thermophilic Coccus
<400>7
atgggcgata?tgaggtactc?cgagctggcc?gaactttaca?gaaggcttga?gaagaccacg?60
ctcaaaacgc?tcaagaccaa?gttcgtcgcg?gacttcctca?agaaaactcc?cgacgatttg?120
ctcgagatag?ttccctacct?gattctcggc?aaggtctttc?cggactggga?cgagcgcgag?180
ctcggcgtcg?gggaaaagtt?gctcataagg?gccgtttcga?tggcaaccgg?cgtccccgag?240
agggaaatcg?agaactcgat?taaggacacg?ggcgatttgg?gcgagagcgt?ggctctggct?300
ctaaagaaga?ggaagcagaa?gagcttcttc?agccagcccc?tcacgataaa?gcgcgtctac?360
agcaccttcg?ttaaggttgc?cgaggcgagc?ggagagggga?gccaggacag?gaagatgaag?420
tacttagcaa?atctcttcat?ggatgctcaa?cccgaggagg?gcaagtacat?agccagaacc?480
gtcctcggaa?cgatgaggac?gggcgtcgcc?gagggaatcc?tgcgcgatgc?catagcggag?540
gccttcaagg?tgaagccaga?gctcgtcgag?agggcctaca?tgctcacgag?cgacttcgga?600
tacgtggcaa?aggttgcaaa?gctcgagggg?aacgaggggc?tctcaaaggt?tagcatacag?660
attgggaagc?cgataaggcc?gatgctagct?caaaacgccg?ccaacgtcaa?ggaagcgcta?720
atcgagatgg?gcggtgaggc?ggccttcgag?attaagtacg?acggtgcgcg?cgttcaggtc?780
caccgcgacg?gggatagggt?gataatctac?tcgaggaggc?tcgagaacgt?cacccgctcg?840
attcctgaga?tagtcgaggc?ggtaaaggcc?tccctgaagc?cttctaaggt?catagtcgag?900
ggcgagctgg?ttgccgtcgg?cgagaacggt?cgcccgaggc?ccttccagta?cgtcctcagg?960
aggttcagga?ggaagtacaa?catcgaggag?atgattgaga?agattccgct?cgagctcaac?1020
ctcttcgata?tcctctacgt?tgatggggag?agtctcatcg?acacgaagtt?cgcagagagg?1080
aggaagaaac?tcgaggagag?tgttgaggag?agcgataaga?taaagctcgc?cgaacagctc?1140
gttacgaaga?aggtcgaaga?ggccgaggag?ttctacaaga?gggcccttga?gctcggccac?1200
gagggcctga?tggcgaagag?gctggactcc?atctacgagc?ccggaaaccg?cggtaagaag?1260
tggctgaaga?ttaagcccac?gatggagaac?cttgacctcg?tcattatcgg?agccgaatgg?1320
ggcgagggga?ggcgcgcgca?cctgcttggc?tcgttcctcg?ttgcggccta?cgaccccgag?1380
agcggtgagt?tcgtcccggt?gggcaaggtc?gggagcggtt?tcaccgatga?agatttggtc?1440
gagttcacca?agatgctcaa?gcccctgatt?gtccgtgaag?agggcaagtt?cgtcgagatt?1500
gagcccaagg?tcgtcatcga?ggtcacctac?caggagatac?agaagagccc?caagtataag?1560
agcggtttcg?cgcttcgctt?cccgcgctac?gtggcgctga?gggaagataa?aagcccggag?1620
gaggctgaca?ccatagagag?ggtcgcccag?ctctacgagc?tccaggagag?gttcaaggcg?1680
aagaagtaat?ctaga 1695
<210>8
<211>564
<212>PRT
＜213〉the unknown
<220>
＜223〉certain strain 9 deg n of thermophilic Coccus
<400>8
Met?Gly?Asp?Met?Arg?Tyr?Ser?Glu?Leu?Ala?Glu?Leu?Tyr?Arg?Arg?Leu
1 5 10 15
Glu?Lys?Thr?Thr?Leu?Lys?Thr?Leu?Lys?Thr?Lys?Phe?Val?Ala?Asp?Phe
20 25 30
Leu?Lys?Lys?Thr?Pro?Asp?Asp?Leu?Leu?Glu?Ile?Val?Pro?Tyr?Leu?Ile
35 40 45
Leu?Gly?Lys?Val?Phe?Pro?Asp?Trp?Asp?Glu?Arg?Glu?Leu?Gly?Val?Gly
50 55 60
Glu?Lys?Leu?Leu?Ile?Arg?Ala?Val?Ser?Met?Ala?Thr?Gly?Val?Pro?Glu
65 70 75 80
Arg?Glu?Ile?Glu?Asn?Ser?Ile?Lys?Asp?Thr?Gly?Asp?Leu?Gly?Glu?Ser
85 90 95
Val?Ala?Leu?Ala?Leu?Lys?Lys?Arg?Lys?Gln?Lys?Ser?Phe?Phe?Ser?Gln
100 105 110
Pro?Leu?Thr?Ile?Lys?Arg?Val?Tyr?Ser?Thr?Phe?Val?Lys?Val?Ala?Glu
115 120 125
Ala?Ser?Gly?Glu?Gly?Ser?Gln?Asp?Arg?Lys?Met?Lys?Tyr?Leu?Ala?Asn
130 135 140
Leu?Phe?Met?Asp?Ala?Gln?Pro?Glu?Glu?Gly?Lys?Tyr?Ile?Ala?Arg?Thr
145 150 155 160
Val?Leu?Gly?Thr?Met?Arg?Thr?Gly?Val?Ala?Glu?Gly?Ile?Leu?Arg?Asp
165 170 175
Ala?Ile?Ala?Glu?Ala?Phe?Lys?Val?Lys?Pro?Glu?Leu?Val?Glu?Arg?Ala
180 185 190
Tyr?Met?Leu?Thr?Ser?Asp?Phe?Gly?Tyr?Val?Ala?Lys?Val?Ala?Lys?Leu
195 200 205
Glu?Gly?Asn?Glu?Gly?Leu?Ser?Lys?Val?Ser?Ile?Gln?Ile?Gly?Lys?Pro
210 215 220
Ile?Arg?Pro?Met?Leu?Ala?Gln?Asn?Ala?Ala?Asn?Val?Lys?Glu?Ala?Leu
225 230 235 240
Ile?Glu?Met?Gly?Gly?Glu?Ala?Ala?Phe?Glu?Ile?Lys?Tyr?Asp?Gly?Ala
245 250 255
Arg?Val?Gln?Val?His?Arg?Asp?Gly?Asp?Arg?Val?Ile?Ile?Tyr?Ser?Arg
260 265 270
Arg?Leu?Glu?Asn?Val?Thr?Arg?Ser?Ile?Pro?Glu?Ile?Val?Glu?Ala?Val
275 280 285
Lys?Ala?Ser?Leu?Lys?Pro?Ser?Lys?Val?Ile?Val?Glu?Gly?Glu?Leu?Val
290 295 300
Ala?Val?Gly?Glu?Asn?Gly?Arg?Pro?Arg?Pro?Phe?Gln?Tyr?Val?Leu?Arg
305 310 315 320
Arg?Phe?Arg?Arg?Lys?Tyr?Asn?Ile?Glu?Glu?Met?Ile?Glu?Lys?Ile?Pro
325 330 335
Leu?Glu?Leu?Asn?Leu?Phe?Asp?Ile?Leu?Tyr?Val?Asp?Gly?Glu?Ser?Leu
340 345 350
Ile?Asp?Thr?Lys?Phe?Ala?Glu?Arg?Arg?Lys?Lys?Leu?Glu?Glu?Ser?Val
355 360 365
Glu?Glu?Ser?Asp?Lys?Ile?Lys?Leu?Ala?Glu?Gln?Leu?Val?Thr?Lys?Lys
370 375 380
Val?Glu?Glu?Ala?Glu?Glu?Phe?Tyr?Lys?Arg?Ala?Leu?Glu?Leu?Gly?His
385 390 395 400
Glu?Gly?Leu?Met?Ala?Lys?Arg?Leu?Asp?Ser?Ile?Tyr?Glu?Pro?Gly?Asn
405 410 415
Arg?Gly?Lys?Lys?Trp?Leu?Lys?Ile?Lys?Pro?Thr?Met?Glu?Asn?Leu?Asp
420 425 430
Leu?Val?Ile?Ile?Gly?Ala?Glu?Trp?Gly?Glu?Gly?Arg?Arg?Ala?His?Leu
435 440 445
Leu?Gly?Ser?Phe?Leu?Val?Ala?Ala?Tyr?Asp?Pro?Glu?Ser?Gly?Glu?Phe
450 455 460
Val?Pro?Val?Gly?Lys?Val?Gly?Ser?Gly?Phe?Thr?Asp?Glu?Asp?Leu?Val
465 470 475 480
Glu?Phe?Thr?Lys?Met?Leu?Lys?Pro?Leu?Ile?Val?Arg?Glu?Glu?Gly?Lys
485 490 495
Phe?Val?Glu?Ile?Glu?Pro?Lys?Val?Val?Ile?Glu?Val?Thr?Tyr?Gln?Glu
500 505 510
Ile?Gln?Lys?Ser?Pro?Lys?Tyr?Lys?Ser?Gly?Phe?Ala?Leu?Arg?Phe?Pro
515 520 525
Arg?Tyr?Val?Ala?Leu?Arg?Glu?Asp?Lys?Ser?Pro?Glu?Glu?Ala?Asp?Thr
530 535 540
Ile?Glu?Arg?Val?Ala?Gln?Leu?Tyr?Glu?Leu?Gln?Glu?Arg?Phe?Lys?Ala
545 550 555 560
Lys?Lys?Ser?Arg
<210>9
<211>564
<212>PRT
＜213〉the unknown
<220>
＜223〉certain strain 9 deg n of thermophilic Coccus
<400>9
Met?Gly?Asp?Met?Arg?Tyr?Ser?Glu?Leu?Ala?Glu?Leu?Tyr?Arg?Arg?Leu
1 5 10 15
Glu?Lys?Thr?Thr?Leu?Lys?Thr?Leu?Lys?Thr?Lys?Phe?Val?Ala?Asp?Phe
20 25 30
Leu?Lys?Lys?Thr?Pro?Asp?Asp?Leu?Leu?Glu?Ile?Val?Pro?Tyr?Leu?Ile
35 40 45
Leu?Gly?Lys?Val?Phe?Pro?Asp?Trp?Asp?Glu?Arg?Glu?Leu?Gly?Val?Gly
50 55 60
Glu?Lys?Leu?Leu?Ile?Arg?Ala?Val?Ser?Met?Ala?Thr?Gly?Val?Pro?Glu
65 70 75 80
Arg?Glu?Ile?Glu?Asn?Ser?Ile?Lys?Asp?Thr?Gly?Asp?Leu?Gly?Glu?Ser
85 90 95
Val?Ala?Leu?Ala?Leu?Lys?Lys?Arg?Lys?Gln?Lys?Ser?Phe?Phe?Ser?Gln
100 105 110
Pro?Leu?Thr?Ile?Lys?Arg?Val?Tyr?Ser?Thr?Phe?Val?Lys?Val?Ala?Glu
115 120 125
Ala?Ser?Gly?Glu?Gly?Ser?Gln?Asp?Arg?Lys?Met?Lys?Tyr?Leu?Ala?Asn
130 135 140
Leu?Phe?Met?Asp?Ala?Gln?Pro?Glu?Glu?Gly?Lys?Tyr?Ile?Ala?Arg?Thr
145 150 155 160
Val?Leu?Gly?Thr?Met?Arg?Thr?Gly?Val?Ala?Glu?Gly?Ile?Leu?Arg?Asp
165 170 175
Ala?Ile?Ala?Glu?Ala?Phe?Lys?Val?Lys?Pro?Glu?Leu?Val?Glu?Arg?Ala
180 185 190
Tyr?Met?Leu?Thr?Ser?Asp?Phe?Gly?Tyr?Val?Ala?Lys?Val?Ala?Lys?Leu
195 200 205
Glu?Gly?Asn?Glu?Gly?Leu?Ser?Lys?Va?lSer?Ile?Gln?Ile?Gly?Lys?Pro
210 215 220
Ile?Arg?Pro?Met?Leu?Ala?Gln?Asn?Ala?Ala?Asn?Val?Lys?Glu?Ala?Leu
225 230 235 240
Ile?Glu?Met?Gly?Gly?Glu?Ala?Ala?Phe?Glu?Ile?Lys?Tyr?Asp?Gly?Ala
245 250 255
Arg?Val?Gln?Val?His?Arg?Asp?Gly?Asp?Arg?ValIle?Ile?Tyr?Ser?Arg
260 265 270
Arg?Leu?Glu?Asn?Val?Thr?Arg?Ser?Ile?Pro?Glu?Ile?Val?Glu?Ala?Val
275 280 285
Lys?Ala?Ser?Leu?Lys?Pro?Ser?Lys?Val?Ile?Val?Glu?Gly?Glu?Leu?Val
290 295 300
Ala?Val?Gly?Glu?Asn?Gly?Arg?Pro?Arg?Pro?Phe?Gln?Tyr?Val?Leu?Arg
305 310 315 320
Arg?Phe?Arg?Arg?Lys?Tyr?Asn?Ile?Glu?Glu?Met?Ile?Glu?Lys?Ile?Pro
325 330 335
Leu?Glu?Leu?Asn?Leu?Phe?Asp?Ile?Leu?Tyr?Val?Asp?Gly?Glu?Ser?Leu
340 345 350
Ile?Asp?Thr?Lys?Phe?Ala?Glu?Arg?Arg?Lys?Lys?Leu?Glu?Glu?Ser?Val
355 360 365
Glu?Glu?Ser?Asp?Lys?Ile?Lys?Leu?Ala?Glu?Gln?Leu?Val?Thr?Lys?Lys
370 375 380
Val?Glu?Glu?Ala?Glu?Glu?Phe?Tyr?Lys?Arg?Ala?Leu?Glu?Leu?Gly?His
385 390 395 400
Glu?Gly?Leu?Met?Ala?Lys?Arg?Leu?Asp?Ser?Ile?Tyr?Glu?Pro?Gly?Asn
405 410 415
Arg?Gly?Lys?Lys?Trp?Leu?Lys?Ile?Lys?Pro?Thr?Met?Glu?Asn?Leu?Asp
420 425 430
Leu?Val?Ile?Ile?Gly?Ala?Glu?Trp?Gly?Glu?Gly?Arg?Arg?Ala?His?Leu
435 440 445
Leu?Gly?Ser?Phe?Leu?Val?Ala?Ala?Tyr?Asp?Pro?Glu?Ser?Gly?Glu?Phe
450 455 460
Val?Pro?Val?Gly?Lys?Val?Gly?Ser?Gly?Phe?Thr?Asp?Glu?Asp?Leu?Val
465 470 475 480
Glu?Phe?Thr?Lys?Met?Leu?Lys?Pro?Leu?Ile?Val?Arg?Glu?Glu?Gly?Lys
485 490 495
Phe?Val?Glu?Ile?Glu?Pro?Lys?Val?Val?Ile?Glu?Val?Thr?Tyr?Gln?Glu
500 505 510
Ile?Gln?Lys?Ser?Pro?Lys?Tyr?Lys?Ser?Gly?Phe?Ala?Leu?Arg?Phe?Pro
515 520 525
Arg?Tyr?Val?Ala?Leu?Arg?Glu?Asp?Lys?Ser?Pro?Glu?Glu?Ala?Asp?Thr
530 535 540
Ile?Glu?Arg?Val?Ala?Gln?Leu?Tyr?Glu?Leu?Gln?Glu?Arg?Phe?Lys?Ala
545 550 555 560
Lys?Lys?Ser?Arg
<210>10
<211>564
<212>PRT
＜213〉the unknown
<220>
＜223〉certain strain 9 deg n of thermophilic Coccus
<400>10
Met?Gly?Asp?Met?Arg?Tyr?Ser?Glu?Leu?Ala?Glu?Leu?Tyr?Arg?Arg?Leu
1 5 10 15
Glu?Lys?Thr?Thr?Leu?Lys?Thr?Leu?Lys?Thr?Lys?Phe?Val?Ala?Asp?Phe
20 25 30
Leu?Lys?Lys?Thr?Pro?Asp?Asp?Leu?Leu?Glu?Ile?Val?Pro?Tyr?Leu?Ile
35 40 45
Leu?Gly?Lys?Val?Phe?Pro?Asp?Trp?Asp?Glu?Arg?Glu?Leu?Gly?Val?Gly
50 55 60
Glu?Lys?Leu?Leu?Ile?Arg?Ala?Val?Ser?Met?Ala?Thr?Gly?Val?Pro?Glu
65 70 75 80
Arg?Glu?Ile?Glu?Asn?Ser?Ile?Lys?Asp?Thr?Gly?Asp?Leu?Gly?Glu?Ser
85 90 95
Val?Ala?Leu?Ala?Leu?Lys?Lys?Arg?Lys?Gln?Lys?Ser?Phe?Phe?Ser?Gln
100 105 110
Pro?Leu?Thr?Ile?Lys?Arg?Val?Tyr?Ser?Thr?Phe?Val?Lys?Val?Ala?Glu
115 120 125
Ala?Ser?Gly?Glu?Gly?Ser?Gln?Asp?Arg?Lys?Met?Lys?Tyr?Leu?Ala?Asn
130 135 140
Leu?Phe?Met?Asp?Ala?Gln?Pro?Glu?Glu?Gly?Lys?Tyr?Ile?Ala?Arg?Thr
145 150 155 160
Val?Leu?Gly?Thr?Met?Arg?Thr?Gly?Val?Ala?Glu?Gly?Ile?Leu?Arg?Asp
165 170 175
Ala?Ile?Ala?Glu?Ala?Phe?Lys?Val?Lys?Pro?Glu?Leu?Val?Glu?Arg?Ala
180 185 190
Tyr?Met?Leu?Thr?Ser?Asp?Phe?Gly?Tyr?Val?Ala?Lys?Val?Ala?Lys?Leu
195 200 205
Glu?Gly?Asn?Glu?Gly?Leu?Ser?Lys?Val?Ser?Ile?Gln?Ile?Gly?Lys?Pro
210 215 220
Ile?Arg?Pro?Met?Leu?Ala?Gln?Asn?Ala?Ala?Asn?Val?Lys?Glu?Ala?Leu
225 230 235 240
Ile?Glu?Met?Gly?Gly?Glu?Ala?Ala?Phe?Glu?Ile?Lys?Tyr?Asp?Gly?Ala
245 250 255
Arg?Val?Gln?Val?His?Arg?Asp?Gly?Asp?Arg?Val?Ile?Ile?Tyr?Ser?Arg
260 265 270
Arg?Leu?Glu?Asn?Val?Thr?Arg?Ser?Ile?Pro?Glu?Ile?Val?Glu?Ala?Val
275 280 285
Lys?Ala?Ser?Leu?Lys?Pro?Ser?Asn?Val?Ile?Val?Glu?Gly?Glu?Leu?Val
290 295 300
Ala?Val?Gly?Glu?Asn?Gly?Arg?Pro?Arg?Pro?Phe?Gln?Tyr?Val?Leu?Arg
305 310 315 320
Arg?Phe?Arg?Arg?Lys?Tyr?Asn?Ile?Glu?Glu?Met?Ile?Glu?Lys?Ile?Pro
325 330 335
Leu?Glu?Leu?Asn?Leu?Phe?Asp?Ile?Leu?Tyr?Val?Glu?Gly?Glu?Ser?Leu
340 345 350
Ile?Asp?Thr?Lys?Phe?Ala?Glu?Arg?Arg?Lys?Lys?Leu?Glu?Glu?Ser?Val
355 360 365
Glu?Glu?Ser?Asp?Lys?Ile?Lys?Leu?Ala?Glu?Gln?Leu?Val?Thr?Lys?Lys
370 375 380
Val?Glu?Glu?Ala?Glu?Glu?Phe?Tyr?Lys?Arg?Ala?Leu?Glu?Leu?Gly?His
385 390 395 400
Glu?Gly?Leu?Met?Ala?Lys?Arg?Leu?Asp?Ser?Ile?Tyr?Glu?Pro?Gly?Asn
405 410 415
Arg?Gly?Lys?Lys?Trp?Leu?Lys?Ile?Lys?Pro?Thr?Met?Glu?Asn?Leu?Asp
420 425 430
Leu?Val?Ile?Ile?Gly?Ala?Glu?Trp?Gly?Glu?Gly?Arg?Arg?Ala?His?Leu
435 440 445
Leu?Gly?Ser?Phe?Leu?Val?Ala?Ala?Tyr?Asp?Pro?Glu?Ser?Gly?Glu?Phe
450 455 460
Val?Pro?Val?Gly?Lys?Val?Gly?Ser?Gly?Phe?Thr?Asp?Glu?Asp?Leu?Val
465 470 475 480
Glu?Phe?Thr?Lys?Met?Leu?Lys?Pro?Leu?Ile?Val?Arg?Glu?Glu?Gly?Lys
485 490 495
Phe?Val?Glu?Ile?Glu?Pro?Lys?Val?Val?Ile?Glu?Val?Thr?Tyr?Gln?Glu
500 505 510
Ile?Gln?Lys?Ser?Pro?Lys?Tyr?Lys?Ser?Gly?Phe?Ala?Leu?Arg?Phe?Pro
515 520 525
Arg?Tyr?Val?Ala?Leu?Arg?Glu?Asp?Lys?Ser?Pro?Glu?Glu?Ala?Asp?Thr
530 535 540
Ile?Glu?Arg?Val?Ala?Gln?Leu?Tyr?Glu?Leu?Gln?Glu?Arg?Phe?Lys?Ala
545 550 555 560
Lys?Lys?Ser?Arg
<210>11
<211>563
<212>PRT
＜213〉the unknown
<220>
＜223〉certain strain 9 deg n of thermophilic Coccus
<400>11
Met?Gly?Asp?Met?Arg?Tyr?Ser?Glu?Leu?Ala?Glu?Leu?Tyr?Arg?Arg?Leu
1 5 10 15
Glu?Lys?Thr?Thr?Leu?Lys?Thr?Leu?Lys?Thr?Lys?Phe?Val?Ala?Asp?Phe
20 25 30
Leu?Lys?Lys?Thr?Pro?Asp?Asp?Leu?Leu?Glu?Ile?Val?Pro?Tyr?Leu?Ile
35 40 45
Leu?Gly?Lys?Val?Phe?Pro?Asp?Trp?Asp?Glu?Arg?Glu?Leu?Gly?Val?Gly
50 55 60
Glu?Lys?Leu?Leu?Ile?Arg?Ala?Val?Ser?Met?Ala?Thr?Gly?Val?Pro?Glu
65 70 75 80
Arg?Glu?Ile?Glu?Asn?Ser?Ile?Lys?Asp?Thr?Gly?Asp?Leu?Gly?Glu?Ser
85 90 95
Val?Ala?Leu?Ala?Leu?Lys?Lys?Arg?Lys?Gln?Lys?Ser?Phe?Phe?Ser?Gln
100 105 110
Pro?Leu?Thr?Ile?Lys?Arg?Val?Tyr?Ser?Thr?Phe?Val?Lys?Val?Ala?Glu
115 120 125
Ala?Ser?Gly?Glu?Gly?Ser?Gln?Arg?Lys?Met?Lys?Tyr?Leu?Ala?Asn?Leu
130 135 140
Phe?Met?Asp?Ala?Gln?Pro?Glu?Glu?Gly?Lys?Tyr?Ile?Ala?Arg?Thr?Val
145 150 155 160
Leu?Gly?Thr?Met?Arg?Thr?Gly?Val?Ala?Glu?Gly?Ile?Leu?Arg?Asp?Ala
165 170 175
Ile?Ala?Glu?Ala?Phe?Lys?Val?Lys?Pro?Glu?Leu?Val?Glu?Arg?Ala?Tyr
180 185 190
Met?Leu?Thr?Ser?Asp?Phe?Gly?Tyr?Val?Ala?Lys?Val?Ala?Lys?Leu?Glu
195 200 205
Gly?Asn?Glu?Gly?Leu?Ser?Lys?Val?Ser?Ile?Gln?Ile?Gly?Lys?Pro?Ile
210 215 220
Arg?Pro?Met?Leu?Ala?Gln?Asn?Ala?Ala?Asn?Val?Lys?Glu?Ala?Leu?Ile
225 230 235 240
Glu?Met?Gly?Gly?Glu?Ala?Ala?Phe?Glu?Ile?Lys?Tyr?Asp?Gly?Ala?Arg
245 250 255
Val?Gln?Val?His?Arg?Asp?Gly?Asp?Arg?Val?Ile?Ile?Tyr?Ser?Arg?Arg
260 265 270
Leu?Glu?Asn?Val?Thr?Arg?Ser?Ile?Pro?Glu?Ile?Val?Glu?Ala?Val?Lys
275 280 285
Ala?Ser?Leu?Lys?Pro?Ser?Asn?Val?Ile?Val?Glu?Gly?Glu?Leu?Val?Ala
290 295 300
Val?Gly?Glu?Asn?Gly?Arg?Pro?Arg?Pro?Phe?Gln?Tyr?Val?Leu?Arg?Arg
305 310 315 320
Phe?Arg?Arg?Lys?Tyr?Asn?Ile?Glu?Glu?Met?Ile?Glu?Lys?Ile?Pro?Leu
325 330 335
Glu?Leu?Asn?Leu?Phe?Asp?Ile?Leu?Tyr?Val?Glu?Gly?Glu?Ser?Leu?Ile
340 345 350
Asp?Thr?Lys?Phe?Ala?Glu?Arg?Arg?Arg?Lys?Leu?Glu?Glu?Ser?Val?Glu
355 360 365
Glu?Gly?Asp?Lys?Ile?Lys?Leu?Ala?Glu?Gln?Leu?Val?Thr?Lys?Lys?Val
370 375 380
Glu?Glu?Ala?Glu?Glu?Phe?Tyr?Lys?Arg?Ala?Leu?Glu?Leu?Gly?His?Glu
385 390 395 400
Gly?Leu?Met?Ala?Lys?Arg?Leu?Asp?Ser?Ile?Tyr?Glu?Pro?Gly?Asn?Arg
405 410 415
Gly?Lys?Lys?Trp?Leu?Lys?Ile?Lys?Pro?Thr?Met?Glu?Asn?Leu?Asp?Leu
420 425 430
Val?Ile?Ile?Gly?Ala?Glu?Trp?Gly?Glu?Gly?Arg?Arg?Ala?His?Leu?Leu
435 440 445
Gly?Ser?Phe?Leu?Val?Ala?Ala?Tyr?Asp?Pro?Glu?Ser?Gly?Glu?Phe?Val
450 455 460
Pro?Val?Gly?Lys?Val?Gly?Ser?Gly?Phe?Thr?Asp?Glu?Asp?Leu?Val?Glu
465 470 475 480
Phe?Thr?Lys?Met?Leu?Lys?Pro?Leu?Ile?Val?Arg?Glu?Glu?Gly?Lys?Phe
485 490 495
Val?Glu?Ile?Glu?Pro?Lys?Val?Val?Ile?Glu?Val?Thr?Tyr?Gln?Glu?Ile
500 505 510
Gln?Lys?Ser?Pro?Lys?Tyr?Lys?Ser?Gly?Phe?Ala?Leu?Arg?Phe?Pro?Arg
515 520 525
Tyr?Val?Ala?Leu?Arg?Glu?Asp?Lys?Ser?Pro?Glu?Glu?Ala?Asp?Thr?Ile
530 535 540
Glu?Arg?Val?Ala?Gln?Leu?Tyr?Glu?Leu?Gln?Glu?Arg?Phe?Lys?Ala?Lys
545 550 555 560
Lys?Ser?Arg
<210>12
<211>564
<212>PRT
＜213〉the unknown
<220>
<223>Thermoccocus?sp.strain?9?degrees?North
<400>12
Met?Gly?Asp?Met?Arg?Tyr?Ser?Glu?Leu?Ala?Glu?Leu?Tyr?Arg?Arg?Leu
1 5 10 15
Glu?Lys?Thr?Thr?Leu?Lys?Thr?Leu?Lys?Thr?Lys?Phe?Val?Ala?Asp?Phe
20 25 30
Leu?Lys?Lys?Thr?Pro?Asp?Asp?Leu?Leu?Glu?Ile?Val?Pro?Tyr?Leu?Ile
35 40 45
Leu?Gly?Lys?Val?Phe?Pro?Asp?Trp?Asp?Glu?Arg?Glu?Leu?Gly?Val?Gly
50 55 60
Glu?Lys?Leu?Leu?Ile?Arg?Ala?Val?Ser?Met?Ala?Thr?Gly?Val?Pro?Glu
65 70 75 80
Arg?Glu?Ile?Glu?Asn?Ser?Ile?Lys?Asp?Thr?Gly?Asp?Leu?Gly?Glu?Ser
85 90 95
Val?Ala?Leu?Ala?Leu?Lys?Lys?Arg?Lys?Gln?Lys?Ser?Phe?Phe?Ser?Gln
100 105 110
Pro?Leu?Thr?Ile?Lys?Arg?Val?Tyr?Ser?Thr?Phe?Val?Lys?Val?Ala?Glu
115 120 125
Ala?Ser?Gly?Glu?Gly?Ser?Gln?Asp?Arg?Lys?Met?Lys?Tyr?Leu?Ala?Asn
130 135 140
Leu?Phe?Met?Asp?Ala?Gln?Pro?Glu?Glu?Gly?Lys?Tyr?Ile?Ala?Arg?Thr
145 150 155 160
Val?Leu?Gly?Thr?Met?Arg?Thr?Gly?Val?Ala?Glu?Gly?Ile?Leu?Arg?Asp
165 170 175
Ala?Ile?Ala?Glu?Ser?Phe?Lys?Val?Lys?Pro?Glu?Leu?Val?Glu?Arg?Ala
180 185 190
Tyr?Met?Leu?Thr?Ser?Asp?Phe?Gly?Tyr?Val?Ala?Lys?Val?Ala?Lys?Leu
195 200 205
Glu?Gly?Asn?Glu?Gly?Leu?Ser?Lys?Val?Ser?Ile?Gln?Ile?Gly?Lys?Pro
210 215 220
Ile?Arg?Pro?Met?Leu?Ala?Gln?Asn?Ala?Ala?Asn?Val?Lys?Glu?Ala?Leu
225 230 235 240
Ile?Glu?Met?Gly?Gly?Glu?Ala?Ala?Phe?Glu?Ile?Lys?Tyr?Asp?Gly?Ala
245 250 255
Arg?Val?Gln?Val?His?Arg?Asp?Gly?Asp?Arg?Val?Ile?Ile?Tyr?Ser?Arg
260 265 270
Arg?Leu?Glu?Asn?Val?Thr?Arg?Ser?Ile?Pro?Glu?Ile?Val?Glu?Ala?Val
275 280 285
Lys?Ala?Ser?Leu?Lys?Pro?Ser?Asn?Val?Ile?Val?Glu?Gly?Glu?Leu?Val
290 295 300
Ala?Val?Gly?Glu?Asn?Gly?Arg?Pro?Arg?Pro?Phe?Gln?Tyr?Val?Leu?Arg
305 310 315 320
Arg?Phe?Arg?Arg?Lys?Tyr?Asn?Ile?Glu?Glu?Met?Ile?Glu?Lys?Ile?Pro
325 330 335
Leu?Glu?Leu?Asn?Leu?Phe?Asp?Ile?Leu?Tyr?Val?Glu?Gly?Glu?Ser?Leu
340 345 350
Ile?Asp?Thr?Lys?Phe?Ala?Glu?Arg?Arg?Lys?Lys?Leu?Glu?Glu?Ser?Val
355 360 365
Glu?Glu?Ser?Asp?Lys?Ile?Lys?Leu?Ala?Glu?Gln?Leu?Val?Thr?Lys?Lys
370 375 380
Val?Glu?Glu?Ala?Glu?Glu?Phe?Tyr?Lys?Arg?Ala?Leu?Glu?Leu?Gly?His
385 390 395 400
Glu?Gly?Leu?Met?Ala?Lys?Arg?Leu?Asp?Ser?Ile?Tyr?Glu?Pro?Gly?Asn
405 410 415
Arg?Gly?Lys?Lys?Trp?Leu?Lys?Ile?Lys?Pro?Thr?Met?Glu?Asn?Leu?Asp
420 425 430
Leu?Val?Ile?Ile?Gly?Ala?Glu?Trp?Gly?Glu?Gly?Arg?Arg?Ala?His?Leu
435 440 445
Leu?Gly?Ser?Phe?Leu?Val?Ala?Ala?Tyr?Asp?Pro?Glu?Ser?Gly?Glu?Phe
450 455 460
Val?Pro?Val?Gly?Lys?Val?Gly?Ser?Gly?Phe?Thr?Asp?Glu?Asp?Leu?Val
465 470 475 480
Glu?Phe?Thr?Lys?Met?Leu?Lys?Pro?Leu?Ile?Val?Arg?Glu?Glu?Gly?Lys
485 490 495
Phe?Val?Glu?Ile?Glu?Pro?Lys?Val?Val?Ile?Glu?Val?Thr?Tyr?Gln?Glu
500 505 510
Ile?Gln?Lys?Ser?Pro?Lys?Tyr?Lys?Ser?Gly?Phe?Ala?Leu?Arg?Phe?Pro
515 520 525
Arg?Tyr?Val?Ala?Leu?Arg?Glu?Asp?Lys?Ser?Pro?Glu?Glu?Ala?Asp?Thr
530 535 540
Ile?Glu?Arg?Val?Ala?Gln?Leu?Tyr?Glu?Leu?Gln?Glu?Arg?Phe?Lys?Ala
545 550 555 560
Lys?Arg?Ser?Arg
<210>13
<211>564
<212>PRT
＜213〉the unknown
<220>
＜223〉certain strain 9 deg n of thermophilic Coccus
<400>13
Met?Gly?Asp?Met?Arg?Tyr?Ser?Glu?Leu?Ala?Glu?Leu?Tyr?Arg?Arg?Leu
1 5 10 15
Glu?Lys?Thr?Thr?Leu?Lys?Thr?Leu?Lys?Thr?Lys?Phe?Val?Ala?Asp?Phe
20 25 30
Leu?Lys?Lys?Thr?Pro?Asp?Asp?Leu?Leu?Glu?Ile?Val?Pro?Tyr?Leu?Ile
35 40 45
Leu?Gly?Lys?Val?Phe?Pro?Asp?Trp?Asp?Glu?Arg?Glu?Leu?Gly?Val?Gly
50 55 60
Glu?Lys?Leu?Leu?Ile?Arg?Ala?Val?Ser?Met?Ala?Thr?Gly?Val?Pro?Glu
65 70 75 80
Arg?Glu?Ile?Glu?Asn?Ser?Ile?Lys?Asp?Thr?Gly?Asp?Leu?Gly?Glu?Ser
85 90 95
Val?Ala?Leu?Ala?Leu?Lys?Lys?Arg?Lys?Gln?Lys?Ser?Phe?Phe?Ser?Gln
100 105 110
Pro?Leu?Thr?Ile?Lys?Arg?Val?Tyr?Ser?Thr?Phe?Val?Lys?Val?Ala?Glu
115 120 125
Ala?Ser?Gly?Glu?Gly?Ser?Gln?Asp?Arg?Lys?Met?Lys?Tyr?Leu?Ala?Asn
130 135 140
Leu?Phe?Met?Asp?Ala?Gln?Pro?Glu?Glu?Gly?Lys?Tyr?Ile?Ala?Arg?Thr
145 150 155 160
Val?Leu?Gly?Thr?Met?Arg?Thr?Gly?Val?Ala?Glu?Gly?Ile?Leu?Arg?Asp
165 170 175
Ala?Ile?Ala?Glu?Ser?Phe?Lys?Val?Lys?Pro?Glu?Leu?Val?Glu?Arg?Ala
180 185 190
Tyr?Met?Leu?Thr?Ser?Asp?Phe?Gly?Tyr?Val?Ala?Lys?Val?Ala?Lys?Leu
195 200 205
Glu?Gly?Asn?Glu?Gly?Leu?Ser?Lys?Val?Ser?Ile?Gln?Ile?Gly?Lys?Pro
210 215 220
Ile?Arg?Pro?Met?Leu?Ala?Gln?Asn?Ala?Ala?Asn?Val?Lys?Glu?Ala?Leu
225 230 235 240
Ile?Glu?Met?Gly?Gly?Glu?Ala?Ala?Phe?Glu?Ile?Lys?Tyr?Asp?Gly?Ala
245 250 255
Arg?Val?Gln?Val?His?Arg?Asp?Gly?Asp?Arg?Val?Ile?Ile?Tyr?Ser?Arg
260 265 270
Arg?Leu?Glu?Asn?Val?Thr?Arg?Ser?Ile?Pro?Glu?Ile?Val?Glu?Ala?Val
275 280 285
Lys?Ala?Ser?Leu?Lys?Pro?Ser?Asn?Val?Ile?Val?Glu?Gly?Glu?Leu?Val
290 295 300
Ala?Val?Gly?Glu?Asn?Gly?Arg?Pro?Arg?Pro?Phe?Gln?Tyr?Val?Leu?Arg
305 310 315 320
Arg?Phe?Arg?Arg?Lys?Tyr?Asn?Ile?Glu?Glu?Met?Ile?Glu?Lys?Ile?Pro
325 330 335
Leu?Glu?Leu?Asn?Leu?Phe?Asp?Ile?Leu?Tyr?Val?Glu?Gly?Glu?Ser?Leu
340 345 350
Ile?Asp?Thr?Lys?Phe?Ala?Glu?Arg?Arg?Lys?Lys?Leu?Glu?Glu?Ser?Val
355 360 365
Glu?Glu?Ser?Asp?Lys?Ile?Lys?Leu?Ala?Glu?Gln?Leu?Val?Thr?Lys?Lys
370 375 380
Val?Glu?Glu?Ala?Glu?Glu?Phe?Tyr?Lys?Arg?Ala?Leu?Glu?Leu?Gly?His
385 390 395 400
Glu?Gly?Leu?Met?Ala?Lys?Arg?Leu?Asp?Ser?Ile?Tyr?Glu?Pro?Gly?Asn
405 410 415
Arg?Gly?Lys?Lys?Trp?Leu?Lys?Ile?Lys?Pro?Thr?Met?Glu?Asn?Leu?Asp
420 425 430
Leu?Val?Ile?Ile?Gly?Ala?Glu?Trp?Gly?Glu?Gly?Arg?Arg?Ala?His?Leu
435 440 445
Leu?Gly?Ser?Phe?Leu?Val?Ala?Ala?Tyr?Asp?Pro?Glu?Ser?Gly?Glu?Phe
450 455 460
Val?Pro?Val?Gly?Lys?Val?Gly?Ser?Gly?Phe?Thr?Asp?Glu?Asp?Leu?Val
465 470 475 480
Glu?Phe?Thr?Lys?Met?Leu?Lys?Pro?Leu?Ile?Val?Arg?Glu?Glu?Gly?Lys
485 490 495
Phe?Val?Glu?Ile?Glu?Pro?Lys?Val?Val?Ile?Glu?Val?Thr?Tyr?Gln?Glu
500 505 510
Ile?Gln?Lys?Ser?Pro?Lys?Tyr?Lys?Ser?Gly?Phe?Ala?Leu?Arg?Phe?Pro
515 520 525
Arg?Tyr?Val?Al?a?Leu?Arg?Glu?Asp?Lys?Ser?Pro?Glu?Glu?Ala?Asp?Thr
530 535 540
lle?Glu?Arg?Val?Ala?Gln?Leu?Tyr?Glu?Leu?Gln?Glu?Arg?Phe?Lys?Ala
545 550 555 560
Lys?Lys?Ser?Arg
<210>14
<211>564
<212>PRT
＜213〉the unknown
<220>
＜223〉certain strain 9 deg n of thermophilic Coccus
<400>14
Met?Gly?Asp?Met?Arg?Tyr?Ser?Glu?Leu?Ala?Glu?Leu?Tyr?Arg?Arg?Leu
1 5 10 15
Glu?Lys?Thr?Thr?Leu?Lys?Thr?Leu?Lys?Thr?Lys?Phe?Val?Ala?Asp?Phe
20 25 30
Leu?Lys?Lys?Thr?Pro?Asp?Asp?Leu?Leu?Glu?Ile?Val?Pro?Tyr?Leu?Ile
35 40 45
Leu?Gly?Lys?Val?Phe?Pro?Asp?Trp?Asp?Glu?Arg?Glu?Leu?Gly?Val?Gly
50 55 60
Glu?Lys?Leu?Leu?Ile?Arg?Ala?Val?Ser?Met?Ala?Thr?Gly?Val?Pro?Glu
65 70 75 80
Arg?Glu?Ile?Glu?Asn?Ser?Ile?Lys?Asp?Thr?Gly?Asp?Leu?Gly?Glu?Ser
85 90 95
Val?Ala?Leu?Ala?Leu?Lys?Lys?Arg?Lys?Gln?Lys?Ser?Phe?Phe?Ser?Gln
100 105 110
Pro?Leu?Thr?Ile?Lys?Arg?Val?Tyr?Ser?Thr?Phe?Val?Lys?Val?Ala?Glu
115 120 125
Ala?Ser?Gly?Glu?Gly?Ser?Gln?Asp?Arg?Lys?Met?Lys?Tyr?Leu?Ala?Asn
130 135 140
Leu?Phe?Met?Asp?Ala?Gln?Pro?Glu?Glu?Gly?Lys?Tyr?Ile?Ala?Arg?Thr
145 150 155 160
Val?Leu?Gly?Thr?Met?Arg?Thr?Gly?Val?Ala?Glu?Gly?Ile?Leu?Arg?Asp
165 170 175
Ala?Ile?Ala?Glu?Ala?Phe?Lys?Val?Lys?Pro?Glu?Leu?Val?Glu?Arg?Ala
180 185 190
Tyr?Met?Leu?Thr?Ser?Asp?Phe?Gly?Tyr?Val?Ala?Lys?Val?Ala?Lys?Leu
195 200 205
Glu?Gly?Asn?Glu?Gly?Leu?Ser?Lys?Val?Ser?Ile?Gln?Ile?Gly?Lys?Pro
210 215 220
Ile?Arg?Pro?Met?Leu?Ala?Gln?Asn?Ala?Ala?Asn?Val?Lys?Glu?Ala?Leu
225 230 235 240
Ile?Glu?Met?Gly?Gly?Glu?Ala?Ala?Phe?Glu?Ile?Lys?Tyr?Asp?Gly?Ala
245 250 255
Arg?Val?Gln?Val?His?Arg?Asp?Gly?Asp?Arg?Val?Ile?Ile?Tyr?Ser?Arg
260 265 270
Arg?Leu?Glu?Asn?Ile?Thr?Arg?Ser?Ile?Pro?Glu?Ile?Val?Glu?Ala?Val
275 280 285
Lys?Ala?Ser?Leu?Lys?Pro?Ser?Lys?Val?Ile?Val?Glu?Gly?Glu?Leu?Val
290 295 300
Ala?Val?Gly?Glu?Asn?Gly?Arg?Pro?Arg?Pro?Phe?Gln?Tyr?Val?Leu?Arg
305 310 315 320
Arg?Phe?Arg?Arg?Lys?Tyr?Asn?Ile?Glu?Glu?Met?Ile?Glu?Lys?Ile?Pro
325 330 335
Leu?Glu?Leu?Asn?Leu?Phe?Asp?Ile?Leu?Tyr?Val?Asp?Gly?Glu?Ser?Leu
340 345 350
Ile?Asp?Thr?Lys?Phe?Ala?Glu?Arg?Arg?Lys?Lys?Leu?Glu?Glu?Ser?Val
355 360 365
Glu?Glu?Ser?Asp?Lys?Ile?Lys?Leu?Ala?Glu?Gln?Leu?Val?Thr?Lys?Lys
370 375 380
Val?Glu?Glu?Ala?Glu?Glu?Phe?Tyr?Lys?Arg?Ala?Leu?Glu?Leu?Gly?His
385 390 395 400
Glu?Gly?Leu?Met?Ala?Lys?Arg?Leu?Asp?Ser?Ile?Tyr?Glu?Pro?Gly?Asn
405 410 415
Arg?Gly?Lys?Lys?Trp?Leu?Lys?Ile?Lys?Pro?Thr?Met?Glu?Asn?Leu?Asp
420 425 430
Leu?Val?Ile?Ile?Gly?Ala?Glu?Trp?Gly?Glu?Gly?Arg?Arg?Ala?His?Leu
435 440 445
Leu?Gly?Ser?Phe?Leu?Val?Ala?Ala?Tyr?Asp?Pro?Glu?Ser?Gly?Glu?Phe
450 455 460
Val?Pro?Val?Gly?Lys?Val?Gly?Ser?Gly?Phe?Thr?Asp?Glu?Asp?Leu?Val
465 470 475 480
Glu?Phe?Thr?Lys?Met?Leu?Lys?Pro?Leu?Ile?Val?Arg?Glu?Glu?Gly?Lys
485 490 495
Phe?Val?Glu?Ile?Glu?Pro?Lys?Val?Val?Ile?Glu?Val?Thr?Tyr?Gln?Glu
500 505 510
Ile?Gln?Lys?Ser?Pro?Lys?Tyr?Lys?Ser?Gly?Phe?Ala?Leu?Arg?Phe?Pro
515 520 525
Arg?Tyr?Val?Ala?Leu?Arg?Glu?Asp?Lys?Ser?Pro?Glu?Glu?Ala?Asp?Thr
530 535 540
Ile?Glu?Arg?Val?Ala?Gln?Leu?Tyr?Glu?Leu?Gln?Glu?Arg?Tyr?Lys?Ala
545 550 555 560
Lys?Arg?Ser?Arg
<210>15
<211>564
<212>PRT
＜213〉the unknown
<220>
＜223〉certain strain 9 deg n of thermophilic Coccus B2
<400>15
Met?Gly?Asp?Met?Arg?Tyr?Ser?Glu?Leu?Ala?Glu?Leu?Tyr?Arg?Arg?Leu
1 5 10 15
Glu?Lys?Thr?Thr?Leu?Lys?Thr?Leu?Lys?Thr?Lys?Phe?Val?Ala?Asp?Phe
20 25 30
Leu?Lys?Lys?Thr?Pro?Asp?Asp?Leu?Leu?Glu?Ile?Val?Pro?Tyr?Leu?Ile
35 40 45
Leu?Gly?Lys?Val?Phe?Pro?Asp?Trp?Asp?Glu?Arg?Glu?Leu?Gly?Val?Gly
50 55 60
Glu?Lys?Leu?Leu?Ile?Arg?Ala?Val?Ser?Met?Ala?Thr?Gly?Val?Pro?Glu
65 70 75 80
Arg?Glu?Ile?Glu?Asn?Ser?Ile?Lys?Asp?Thr?Gly?Asp?Leu?Gly?Glu?Ser
85 90 95
Val?Ala?Leu?Ala?Leu?Lys?Lys?Arg?Lys?Gln?Lys?Ser?Phe?Phe?Ser?Gln
100 105 110
Pro?Leu?Thr?Ile?Lys?Arg?Val?Tyr?Ser?Thr?Phe?Val?Lys?Val?Ala?Glu
115 120 125
Ala?Ser?Gly?Glu?Gly?Ser?Gln?Asp?Arg?Lys?Met?Lys?Tyr?Leu?Ala?Asn
130 135 140
Leu?Phe?Met?Asp?Ala?Gln?Pro?Glu?Glu?Gly?Lys?Tyr?Ile?Ala?Arg?Thr
145 150 155 160
Val?Leu?Gly?Thr?Met?Arg?Thr?Gly?Val?Ala?Glu?Gly?Ile?Leu?Arg?Asp
165 170 175
Ala?Ile?Ala?Glu?Ser?Phe?Lys?Val?Lys?Pro?Glu?Leu?Val?Glu?Arg?Ala
180 185 190
Tyr?Met?Leu?Thr?Ser?Asp?Phe?Gly?Tyr?Val?Ala?Lys?Val?Ala?Lys?Leu
195 200 205
Glu?Gly?Asn?Glu?Gly?Leu?Ser?Lys?Val?Ser?Ile?Gln?Ile?Gly?Lys?Pro
210 215 220
Ile?Arg?Pro?Met?Leu?Ala?Gln?Asn?Ala?Ala?Asn?Val?Lys?Glu?Ala?Leu
225 230 235 240
Ile?Glu?Met?Gly?Gly?Glu?Ala?Ala?Phe?Glu?Ile?Lys?Tyr?Asp?Gly?Ala
245 250 255
Arg?Val?Gln?Val?His?Arg?Asp?Gly?Asp?Arg?Val?Ile?Ile?Tyr?Ser?Arg
260 265 270
Arg?Leu?Glu?Asn?Val?Thr?Arg?Ser?Ile?Pro?Glu?Ile?Val?Glu?Ala?Val
275 280 285
Lys?Ala?Ser?Leu?Lys?Pro?Ser?Asn?Val?Ile?Val?Glu?Gly?Glu?Leu?Val
290 295 300
Ala?Val?Gly?Glu?Asn?Gly?Arg?Pro?Arg?Pro?Phe?Gln?Tyr?Val?Leu?Arg
305 310 315 320
Arg?Phe?Arg?Arg?Lys?Tyr?Asn?Ile?Glu?Glu?Met?Ile?Glu?Lys?Ile?Pro
325 330 335
Leu?Glu?Leu?Asn?Leu?Phe?Asp?Ile?Leu?Tyr?Val?Glu?Gly?Glu?Ser?Leu
340 345 350
Ile?Asp?Thr?Lys?Phe?Ala?Glu?Arg?Arg?Lys?Lys?Leu?Glu?Glu?Ser?Val
355 360 365
Glu?Glu?Ser?Asp?Lys?Ile?Lys?Leu?Ala?Glu?Gln?Leu?Val?Thr?Lys?Lys
370 375 380
Val?Glu?Glu?Ala?Glu?Glu?Phe?Tyr?Lys?Arg?Ala?Leu?Glu?Leu?Gly?His
385 390 395 400
Glu?Gly?Leu?Met?Ala?Lys?Arg?Leu?Asp?Ser?Ile?Tyr?Glu?Pro?Gly?Asn
405 410 415
Arg?Gly?Lys?Lys?Trp?Leu?Lys?Ile?Lys?Pro?Thr?Met?Glu?Asn?Leu?Asp
420 425 430
Leu?Val?Ile?Ile?Gly?Ala?Glu?Trp?Gly?Glu?Gly?Arg?Arg?Ala?His?Leu
435 440 445
Leu?Gly?Ser?Phe?Leu?Val?Ala?Ala?Tyr?Asp?Pro?Glu?Ser?Gly?Glu?Phe
450 455 460
Val?Pro?Val?Gly?Lys?Val?Gly?Ser?Gly?Phe?Thr?Asp?Glu?Asp?Leu?Val
465 470 475 480
Glu?Phe?Thr?Lys?Met?Leu?Lys?Pro?Leu?Ile?Val?Arg?Glu?Glu?Gly?Lys
485 490 495
Phe?Val?Glu?Ile?Glu?Pro?Lys?Val?Val?Ile?Glu?Val?Thr?Tyr?Gln?Glu
500 505 510
Ile?Gln?Lys?Ser?Pro?Lys?Tyr?Lys?Ser?Gly?Phe?Ala?Leu?Arg?Phe?Pro
515 520 525
Arg?Tyr?Val?Ala?Leu?Arg?Glu?Asp?Lys?Ser?Pro?Glu?Glu?Ala?Asp?Thr
530 535 540
Ile?Glu?Arg?Val?Ala?Gln?Leu?Tyr?Glu?Leu?Gln?Glu?Arg?Phe?Lys?Ala
545 550 555 560
Lys?Lys?Ser?Arg
<210>16
<211>559
<212>PRT
＜213〉the unknown
<220>
＜223〉the happiness thermophilic coccus of cigarette (Thermococcus sp.tfumicolans)
<400>16
Met?Lys?Tyr?Ser?Glu?Leu?Ala?Gly?Leu?Tyr?Arg?Arg?Leu?Glu?Lys?Thr
1 5 10 15
Thr?Leu?Lys?Thr?Leu?Lys?Thr?Arg?Phe?Val?Ala?Asp?Phe?Leu?Lys?Asn
20 25 30
Val?Pro?Asp?Glu?Leu?Leu?Glu?Ile?Val?Pro?Tyr?Leu?Ile?Leu?Gly?Lys
35 40 45
Val?Phe?Pro?Asp?Trp?Asp?Glu?Arg?Glu?Leu?Gly?Val?Gly?Glu?Lys?Leu
50 55 60
Leu?Ile?Lys?Ala?Val?Ser?Ile?Ala?Thr?Gly?Val?Pro?Glu?Gly?Glu?Ile
65 70 75 80
Glu?Asn?Ser?Ile?Lys?Asp?Thr?Gly?Asp?Leu?Gly?Glu?Ser?Ile?Ala?Leu
85 90 95
Ala?Val?Lys?Lys?Lys?Lys?Gln?Lys?Ser?Phe?Phe?Ser?Gln?Pro?Leu?Thr
100 105 110
Ile?Lys?Arg?Val?Tyr?Asp?Thr?Phe?ValLys?Val?Ala?Glu?Ser?Gln?Gly
115 120 125
Glu?Gly?Ser?Gln?Asp?Arg?Lys?Met?Lys?Tyr?Leu?Ala?Asn?Leu?Phe?Met
130 135 140
Asp?Ala?Gln?Pro?Glu?Glu?Ala?Lys?Tyr?Ile?Ala?Arg?Thr?Val?Leu?Gly
145 150 155 160
Thr?Met?Arg?Thr?Gly?Val?Ala?Glu?Gly?Ile?Leu?Arg?Asp?Ala?Ile?Ala
165 170 175
Glu?Ala?Phe?Lys?Val?Lys?Ala?Glu?Leu?Val?Glu?Arg?Ala?Tyr?Met?Leu
180 185 190
Thr?Ser?Asp?Phe?Gly?Tyr?Val?Thr?Lys?Val?Ala?Lys?Leu?Glu?Gly?Asn
195 200 205
Glu?Gly?Leu?Ser?Lys?Val?Arg?Ile?Gln?Val?Gly?Lys?Pro?Val?Arg?Pro
210 215 220
Met?Leu?Ala?Gln?Asn?Ala?Ala?Ser?Val?Lys?Asp?Ala?Leu?Leu?Glu?Met
225 230 235 240
Gly?Gly?Glu?Ala?Ala?Phe?Glu?Ile?Lys?Tyr?Asp?Gly?Ala?Arg?Val?Gln
245 250 255
Val?His?Lys?Asp?Gly?Asp?Arg?Val?Val?Ile?Tyr?Ser?Arg?Arg?Leu?Glu
260 265 270
Asn?Val?Thr?Arg?Ser?Ile?Pro?Glu?Ile?Val?Glu?Ala?Val?Arg?Ser?Gln
275 280 285
Leu?Arg?Pro?Glu?Lys?Ala?Ile?Val?Glu?Gly?Glu?Leu?ValAla?Val?Gly
290 295 300
Asp?Gly?Gly?Lys?Pro?Arg?Pro?Phe?Gln?Tyr?Val?Leu?Arg?Arg?Phe?Arg
305 310 315 320
Arg?Lys?Tyr?Asn?Ile?Glu?Glu?Met?Ile?Glu?Arg?Ile?Pro?Leu?Glu?Leu
325 330 335
Asn?Leu?Phe?Asp?Val?Leu?Tyr?Val?Asp?Gly?Glu?Ser?Leu?Val?Asp?Thr
340 345 350
Pro?Phe?Met?Glu?Arg?Arg?Lys?Arg?Leu?Glu?Glu?Ala?Val?Glu?Glu?Ser
355 360 365
Glu?Arg?Ile?Lys?Leu?Ala?Gln?Gln?Leu?Val?Thr?Lys?Lys?Ala?Glu?Glu
370 375 380
Ala?Glu?Glu?Phe?Tyr?Arg?Arg?Ala?Leu?Glu?Leu?Gly?His?Glu?Gly?Leu
385 390 395 400
Met?Ala?Lys?Arg?Leu?Asp?Ser?Val?Tyr?Glu?Pro?Gly?Asn?Arg?Gly?Lys
405 410 415
Lys?Trp?Leu?Lys?Ile?Lys?Pro?Thr?Met?Glu?Asp?Leu?Asp?Leu?Val?Ile
420 425 430
Ile?Gly?Ala?Glu?Trp?Gly?Glu?Gly?Arg?Arg?Ala?Hi?s?Leu?Leu?Gly?Ser
435 440 445
Phe?Leu?Val?Ala?Ala?Tyr?Asp?Gln?His?Arg?Gly?Glu?Phe?Val?Pro?Val
450 455 460
Gly?Lys?Val?Gly?Ser?Gly?Phe?Thr?Asp?Glu?Asp?Leu?Ala?Glu?Phe?Thr
465 470 475 480
Lys?Met?Leu?Lys?Pro?Leu?Ile?Val?Arg?Glu?Glu?Gly?Lys?Tyr?Val?Glu
485 490 495
Ile?Glu?Pro?Arg?Val?Val?Ile?Gln?Val?Thr?Tyr?Gln?Glu?Ile?Gln?Lys
500 505 510
Ser?Pro?Lys?Tyr?Glu?Ser?Gly?Phe?Ala?Leu?Arg?Phe?Pro?Arg?Tyr?Val
515 520 525
Ala?Leu?Arg?Glu?Asp?Lys?Ser?Pro?Glu?Glu?Ala?Asp?Thr?Ile?Glu?Arg
530 535 540
Ile?Ser?Glu?Leu?Tyr?Gly?Leu?Gln?Glu?Arg?Phe?Lys?Ala?Lys?Arg
545 550 555
<210>17
<211>562
<212>PRT
＜213〉the unknown
<220>
＜223〉the thermophilic coccus of Ke Dakala (Thermococcus sp.tkodakaraenis)
<400>17
Met?Ser?Asp?Met?Arg?Tyr?Ser?Glu?Leu?Ala?Asp?Leu?Tyr?Arg?Arg?Leu
1 5 10 15
Glu?Lys?Thr?Thr?Leu?Lys?Thr?Leu?Lys?Thr?Lys?Phe?Val?Ala?Asp?Phe
20 25 30
Leu?Lys?Lys?Thr?Pro?Asp?Glu?Leu?Leu?Glu?Ile?Val?Pro?Tyr?Leu?Ile
35 40 45
Leu?Gly?Lys?Val?Phe?Pro?Asp?Trp?Asp?Glu?Arg?Glu?Leu?Gly?Val?Gly
50 55 60
Glu?Lys?Leu?Leu?Ile?Lys?Ala?Val?Ser?Met?Ala?Thr?Gly?Val?Pro?Glu
65 70 75 80
Lys?Glu?Ile?Glu?Asp?Ser?Val?Arg?Asp?Thr?Gly?Asp?Leu?Gly?Glu?Ser
85 90 95
Val?Ala?Leu?Ala?Ile?Lys?Lys?Lys?Lys?Gln?Lys?Ser?Phe?Phe?Ser?Gln
100 105 110
Pro?Leu?Thr?Ile?Lys?Arg?Val?Tyr?Asp?Thr?Phe?Val?Lys?Ile?Ala?Glu
115 120 125
Ala?Gln?Gly?Glu?Gly?Ser?Gln?Asp?Arg?Lys?Met?Lys?Tyr?Leu?Ala?Asn
130 135 140
Leu?Phe?Met?Asp?Ala?Glu?Pro?Glu?Glu?Gly?Lys?Tyr?Leu?Ala?Arg?Thr
145 150 155 160
Val?Leu?Gly?Thr?Met?Arg?Thr?Gly?Val?Ala?Glu?Gly?Ile?Leu?Arg?Asp
165 170 175
Ala?Ile?Ala?Glu?Ala?Phe?Arg?Val?Lys?Pro?Glu?Leu?Val?Glu?Arg?Ala
180 185 190
Tyr?Met?Leu?Thr?Ser?Asp?Phe?Gly?Tyr?Val?Ala?Lys?Ile?Ala?Lys?Leu
195 200 205
Glu?Gly?Asn?Glu?Gly?Leu?Ser?Lys?Val?ArgIle?Gln?Ile?Gly?Lys?Pro
210 215 220
Ile?Arg?Pro?Met?Leu?Ala?Gln?Asn?Ala?Ala?Ser?Val?Lys?Asp?Ala?Leu
225 230 235 240
Ile?Glu?Met?Gly?Gly?Glu?Ala?Ala?Phe?Glu?Ile?Lys?Tyr?Asp?Gly?Ala
245 250 255
Arg?Val?Gln?Val?His?Lys?Asp?Gly?Asp?Lys?Val?Ile?Val?Tyr?Ser?Arg
260 265 270
Arg?Leu?Glu?Asn?Val?Thr?Arg?Ser?Ile?Pro?Glu?Val?Ile?Glu?Ala?Ile
275 280 285
Lys?Ala?Ala?Leu?Lys?Pro?Glu?Lys?Ala?Ile?Val?Glu?Gly?Glu?Leu?Val
290 295 300
Ala?Val?Gly?Glu?Asn?Gly?Arg?Pro?Arg?Pro?Phe?Gln?Tyr?Val?Leu?Arg
305 310 315 320
Arg?Phe?Arg?Arg?Lys?Tyr?Asn?Ile?Asp?Glu?Met?Ile?Glu?Lys?Ile?Pro
325 330 335
Leu?Glu?Leu?Asn?Leu?Phe?Asp?Val?Met?Phe?Val?Asp?Gly?Glu?Ser?Leu
340 345 350
Ile?Glu?Thr?Lys?Phe?Ile?Asp?Arg?Arg?Asn?Lys?Leu?Glu?Glu?Ile?Val
355 360 365
Lys?Glu?Ser?Glu?Lys?Ile?Lys?Leu?Ala?Glu?Gln?Leu?Ile?Thr?Lys?Lys
370 375 380
Val?Glu?Glu?Ala?Glu?Ala?Phe?TyrArg?Arg?Ala?Leu?Glu?Leu?Gly?His
385 390 395 400
Glu?Gly?Leu?Met?Ala?Lys?Arg?Leu?Asp?Ser?Ile?Tyr?Glu?Pro?Gly?Asn
405 410 415
Arg?Gly?Lys?Lys?Trp?Leu?Lys?Ile?Lys?Pro?Thr?Met?Glu?Asn?Leu?Asp
420 425 430
Leu?Val?Ile?Ile?Gly?Ala?Glu?Trp?Gly?Glu?Gly?Arg?Arg?Ala?His?Leu
435 440 445
Leu?Gly?Ser?Phe?Leu?Val?Ala?Ala?Tyr?Asp?Pro?His?Ser?Gly?Glu?Phe
450 455 460
Leu?Pro?Val?Gly?Lys?Val?Gly?Ser?Gly?Phe?Thr?Asp?Glu?Asp?Leu?Val
465 470 475 480
Glu?Phe?Thr?Lys?Met?Leu?Lys?Pro?Tyr?Ile?Val?Arg?Gln?Glu?Gly?Lys
485 490 495
Phe?Val?Glu?Ile?Glu?Pro?Lys?Phe?Val?Ile?Glu?Val?Thr?Tyr?Gln?Glu
500 505 510
Ile?Gln?Lys?Ser?Pro?Lys?Tyr?Lys?Ser?Gly?Phe?Ala?Leu?Arg?Phe?Pro
515 520 525
Arg?Tyr?Val?Ala?Leu?Arg?Glu?Asp?Lys?Ser?Pro?Glu?Glu?Ala?Asp?Thr
530 535 540
Ile?Glu?Arg?Val?Ala?Glu?Leu?Tyr?Glu?Leu?Gln?Glu?Arg?Phe?Lys?Ala
545 550 555 560
Lys?Lys
<210>18
<211>559
<212>PRT
＜213〉the unknown
<220>
＜223〉deep-sea hot-bulb bacterium (Pyrococcus sp.pabyssi)
<400>18
Met?Arg?Tyr?Ile?Glu?Leu?Ala?Gln?Leu?Tyr?Gln?Lys?Leu?Glu?Lys?Thr
1 5 10 15
Thr?Met?Lys?Leu?Ile?Lys?Thr?Arg?Leu?Val?Ala?Asp?Phe?Leu?Lys?Lys
20 25 30
Val?Pro?Glu?Asp?His?Leu?Glu?Phe?Ile?Pro?Tyr?Leu?Ile?Leu?Gly?Asp
35 40 45
Val?Phe?Pro?Glu?Trp?Asp?Glu?Arg?Glu?Leu?Gly?Val?Gly?Glu?Lys?Leu
50 55 60
Leu?Ile?Lys?Ala?Val?Ser?Met?Ala?Thr?Gly?Ile?Asp?Ser?Lys?Glu?Ile
65 70 75 80
Glu?Asn?Ser?Val?Lys?Asp?Thr?Gly?Asp?Leu?Gly?Glu?Ser?Ile?Ala?Leu
85 90 95
Ala?Val?Lys?Arg?Arg?Lys?Gln?Lys?Ser?Phe?Phe?Ser?Gln?Pro?Leu?Thr
100 105 110
Ile?Lys?Arg?Val?Tyr?Gln?Thr?Leu?Val?Lys?Val?Ala?Glu?Thr?Thr?Gly
115 120 125
Glu?Gly?Ser?Gln?Asp?Lys?Lys?Met?Lys?Tyr?Leu?Ala?Asn?Leu?Phe?Met
130 135 140
Asp?Ala?Glu?Pro?Ile?Glu?Ala?Lys?Tyr?Ile?Ala?Arg?Thr?Val?Leu?Gly
145 150 155 160
Thr?Met?Arg?Thr?Gly?Val?Ala?Glu?Gly?Leu?Leu?Arg?Asp?Ala?Ile?Ser
165 170 175
Leu?Ala?Phe?Asn?Val?Lys?Val?Glu?Leu?Val?Glu?Arg?Ala?Tyr?Met?Leu
180 185 190
Thr?Ser?Asp?Phe?Gly?Phe?Val?Ala?Lys?Ile?Ala?Lys?Thr?Glu?Gly?Asn
195 200 205
Asp?Gly?Leu?Ala?Lys?Val?Thr?Ile?Gln?Ile?Gly?Lys?Pro?Ile?Lys?Pro
210 215 220
Met?Leu?Ala?Gln?Gln?Ala?Ala?Asn?Ile?Lys?Glu?Ala?Leu?Leu?Glu?Met
225 230 235 240
Gly?Gly?Glu?Ala?Glu?Phe?Glu?Ile?Lys?Tyr?Asp?Gly?Ala?Arg?Val?Gln
245 250 255
Val?His?Lys?Asp?Gly?Glu?Lys?Val?Thr?Ile?Tyr?Ser?Arg?Arg?Leu?Glu
260 265 270
Asn?Val?Thr?Arg?Ala?Ile?Pro?Glu?Ile?Val?Glu?Ala?Ile?Lys?Glu?Ala
275 280 285
Leu?Lys?Pro?Ala?Lys?Ala?Ile?Val?Glu?Gly?Glu?Leu?Val?Ala?Ile?Gly
290 295 300
Glu?Asp?Gly?Arg?Pro?Leu?Pro?Phe?Gln?Tyr?Val?Leu?Arg?Arg?Phe?Arg
305 310 315 320
Arg?Lys?Tyr?Asn?Ile?Glu?Glu?Met?Met?Glu?Lys?Ile?Pro?Leu?Glu?Leu
325 330 335
Asn?Leu?Phe?Asp?Val?Leu?Tyr?Val?Asp?Gly?Val?Ser?Leu?Ile?Asp?Thr
340 345 350
Lys?Phe?Met?Glu?Arg?Arg?Lys?Lys?Leu?Glu?Glu?Ile?Val?Glu?Ala?Asn
355 360 365
Gly?Lys?Val?Lys?Ile?Ala?Glu?Asn?Leu?Ile?Thr?Lys?Asn?Val?Glu?Glu
370 375 380
Ala?Glu?Gln?Phe?Tyr?Lys?Arg?Ala?Leu?Glu?Met?Gly?His?Glu?Gly?Leu
385 390 395 400
Met?Ala?Lys?Arg?Leu?Asp?Ala?Ile?Tyr?Glu?Pro?Gly?Asn?Arg?Gly?Lys
405 410 415
Lys?Trp?Leu?Lys?Ile?Lys?Pro?Thr?Met?Glu?Asn?Leu?Asp?Leu?Val?Ile
420 425 430
Ile?Gly?Ala?Glu?Trp?Gly?Glu?Gly?Arg?Arg?Ala?His?Leu?Leu?Gly?Ser
435 440 445
Phe?Ile?Leu?Gly?Ala?Tyr?Asp?Pro?Glu?Thr?Gly?Glu?Phe?Leu?Glu?Val
450 455 460
Gly?Lys?Val?Gly?Ser?Gly?Phe?Thr?Asp?Asp?Asp?Leu?Val?Glu?Phe?Thr
465 470 475 480
Lys?Met?Leu?Arg?Pro?Leu?Ile?Ile?Lys?Glu?Glu?Gly?Lys?Arg?Val?Trp
485 490 495
Ile?Gln?Pro?Lys?Val?Val?Ile?Glu?Val?Thr?Tyr?Gln?Glu?Ile?Gln?Lys
500 505 510
Ser?Pro?Lys?Tyr?Arg?Ser?Gly?Phe?Ala?Leu?Arg?Phe?Pro?Arg?Tyr?Val
515 520 525
Ala?Leu?Arg?Glu?Asp?Lys?Gly?Pro?Glu?Asp?Ala?Asp?Thr?Ile?Glu?Arg
530 535 540
Ile?Ala?Gln?Leu?Tyr?Glu?Leu?Gln?Glu?Arg?Met?Lys?Gly?Lys?Val
545 550 555
<210>19
<211>561
<212>PRT
＜213〉the unknown
<220>
＜223〉fierce hot-bulb bacterium (Pyrococcus sp.pfuriosus)
<400>19
Met?Arg?Tyr?Leu?Glu?Leu?Ala?Gln?Leu?Tyr?Gln?Lys?Leu?Glu?Lys?Thr
1 5 10 15
Thr?Met?Lys?Leu?Ile?Lys?Thr?Arg?Leu?Val?Ala?Asp?Phe?Leu?Lys?Lys
20 25 30
Val?Pro?Asp?Asp?His?Leu?Glu?Phe?Ile?Pro?Tyr?Leu?Ile?Leu?Gly?Glu
35 40 45
Val?Phe?Pro?Glu?Trp?Asp?Glu?Arg?Glu?Leu?Gly?Val?Gly?Glu?Lys?Leu
50 55 60
Leu?Ile?Lys?Ala?Val?Ala?Met?Ala?Thr?Gly?Ile?Asp?Ala?Lys?Glu?Ile
65 70 75 80
Glu?Glu?Ser?Val?Lys?Asp?Thr?Gly?Asp?Leu?Gly?Glu?Ser?Ile?Ala?Leu
85 90 95
Ala?Val?Lys?Lys?Lys?Lys?Gln?Lys?Ser?Phe?Phe?Ser?Gln?Pro?Leu?Thr
100 105 110
Ile?Lys?Arg?Val?Tyr?Gln?Thr?Leu?Val?Lys?Val?Ala?Glu?Thr?Thr?Gly
115 120 125
Glu?Gly?Ser?Gln?Asp?Lys?Lys?Val?Lys?Tyr?Leu?Ala?Asp?Leu?Phe?Met
130 135 140
Asp?Ala?Glu?Pro?Leu?Glu?Ala?Lys?Tyr?Leu?Ala?Arg?Thr?Ile?Leu?Gly
145 150 155 160
Thr?Met?Arg?Thr?Gly?Val?Ala?Glu?Gly?Leu?Leu?Arg?Asp?Ala?Ile?Ala
165 170 175
Met?Ala?Phe?His?Val?Lys?Val?Glu?Leu?Val?Glu?Arg?Ala?Tyr?Met?Leu
180 185 190
Thr?Ser?Asp?Phe?Gly?Tyr?Val?Ala?Lys?Ile?Ala?Lys?Leu?Glu?Gly?Asn
195 200 205
Glu?Gly?Leu?Ala?Lys?Val?Gln?Val?Gln?Leu?Gly?Lys?Pro?Ile?Lys?Pro
210 215 220
Met?Leu?Ala?Gln?Gln?Ala?Ala?Ser?Ile?Arg?Asp?Ala?Leu?Leu?Glu?Met
225 230 235 240
Gly?Gly?Glu?Ala?Glu?Phe?Glu?Ile?Lys?Tyr?Asp?Gly?Ala?Arg?Val?Gln
245 250 255
Val?His?Lys?Asp?Gly?Ser?Lys?Ile?Ile?Val?Tyr?Ser?Arg?Arg?Leu?Glu
260 265 270
Asn?Val?Thr?Arg?Ala?Ile?Pro?Glu?Ile?Val?Glu?Ala?Leu?Lys?Glu?Ala
275 280 285
Ile?Ile?Pro?Glu?Lys?Ala?Ile?Val?Glu?Gly?Glu?Leu?Val?Ala?Ile?Gly
290 295 300
Glu?Asn?Gly?Arg?Pro?Leu?Pro?Phe?Gln?Tyr?Val?Leu?Arg?Arg?Phe?Arg
305 310 315 320
Arg?Lys?His?Asn?Ile?Glu?Glu?Met?Met?Glu?Lys?Ile?Pro?Leu?Glu?Leu
325 330 335
Asn?Leu?Phe?Asp?Val?Leu?Tyr?Val?Asp?Gly?Gln?Ser?Leu?Ile?Asp?Thr
340 345 350
Lys?Phe?Ile?Asp?Arg?Arg?Arg?Thr?Leu?Glu?Glu?Ile?Ile?Lys?Gln?Asn
355 360 365
Glu?Lys?Ile?Lys?Val?Ala?Glu?Asn?Leu?Ile?Thr?Lys?Lys?Val?Glu?Glu
370 375 380
Ala?Glu?Ala?Phe?Tyr?Lys?Arg?Ala?Leu?Glu?Met?Gly?His?Glu?Gly?Leu
385 390 395 400
Met?Ala?Lys?Arg?Leu?Asp?Ala?Val?Tyr?Glu?Pro?Gly?Asn?Arg?Gly?Lys
405 410 415
Lys?Trp?Leu?Lys?Ile?Lys?Pro?Thr?Met?Glu?Asn?Leu?Asp?Leu?Val?Ile
420 425 430
Ile?Gly?Ala?Glu?Trp?Gly?Glu?Gly?Arg?Arg?Ala?His?Leu?Phe?Gly?Ser
435 440 445
Phe?Ile?Leu?Gly?Ala?Tyr?Asp?Pro?Glu?Thr?Gly?Glu?Phe?Leu?Glu?Val
450 455 460
Gly?Lys?Val?Gly?Ser?Gly?Phe?Thr?Asp?Asp?Asp?Leu?Val?Glu?Phe?Thr
465 470 475 480
Lys?Met?Leu?Lys?Pro?Leu?Ile?Ile?Lys?Glu?Glu?Gly?Lys?Arg?Val?Trp
485 490 495
Leu?Gln?Pro?Lys?Val?Val?Ile?Glu?Val?Thr?Tyr?Gln?Glu?Ile?Gln?Lys
500 505 510
Ser?Pro?Lys?Tyr?Arg?Ser?Gly?Phe?Ala?Leu?Arg?Phe?Pro?Arg?Phe?Val
515 520 525
Ala?Leu?Arg?Asp?Asp?Lys?Gly?Pro?Glu?Asp?Ala?Asp?Thr?Ile?Glu?Arg
530 535 540
Ile?Ala?Gln?Leu?Tyr?Glu?Leu?Gln?Glu?Lys?Met?Lys?Gly?Lys?Val?Glu
545 550 555 560
Ser
<210>20
<211>31
<212>DNA
＜213〉the unknown
<220>
＜223〉primer
<220>
<221>misc_feature
<222>(15)..(15)
<223>r＝g?or?a
<220>
<221>misc_feature
<222>(20)..(20)
<223>y＝t/u?or?c
<220>
<221>misc_feature
<222>(24)..(24)
<223>m＝a?or?c
<220>
<221>misc_feature
<222>(25)..(25)
<223>r＝g?or?a
<220>
<221>misc_feature
<222>(26)..(26)
<223>s＝g?or?c
<400>20
cggtggtgca?tatgrgcgay?atgmrstact?c 31
<210>21
<211>40
<212>DNA
＜213〉the unknown
<220>
＜223〉primer
<220>
<221>misc_feature
<222>(17)..(17)
<223>y＝t/u?or?c
<400>21
ataaactcta?gattacytct?tcgccttgaa?cctctcctgg 40
<210>22
<211>39
<212>DNA
＜213〉the unknown
<220>
＜223〉primer
<220>
<221>misc_feature
<222>(20)..(20)
<223>y＝t/u?or?c
<400>22
cggtggtgca?tatgggcgay?atgaggtact?ccgagctgg 39
<210>23
<211>23
<212>DNA
＜213〉the unknown
<220>
＜223〉primer
<400>23
cgaacgtcgc?gcagagaaac?agg 23
<210>24
<211>20
<212>DNA
＜213〉the unknown
<220>
＜223〉primer
<400>24
cctgctctgc?cgcttcacgc 20
Claims (11)
1. pure basically recombinant protein, it has the dna ligase activity, and has at least 91% amino acid sequence identity with SEQID NO:13.
2. pure basically protein, it has the dna ligase activity, by being selected from following dna sequence encoding:
(a) with the substantially the same sequence of SEQ ID NO:2;
(b) with SEQ ID NO:2 complementary sequence basically;
(c) under stringent condition, with the sequence of SEQ ID NO:2 hybridization; With
(d) sequence of coding SEQ ID NO:13.
3. protein according to claim 1 wherein after 30 minutes, has kept at least 25% ligase enzyme activity at incubation under about 100 ℃ temperature.
4. protein according to claim 2 wherein after 30 minutes, has kept at least 25% ligase enzyme activity at incubation under about 100 ℃ temperature.
5. according to claim 1,2,3 or 4 described protein, it utilizes ATP rather than NAD in connection procedure
+As cofactor.
6. the DNA of coding DNA ligase enzyme, described DNA have and are selected from following sequence:
(a) with the substantially the same sequence of SEQ ID NO:2;
(b) with SEQ ID NO:2 complementary sequence basically;
(c) under stringent condition, with the sequence of SEQ ID NO:2 hybridization; With
(d) sequence of coding SEQ ID NO:13.
7. carrier contains the described DNA of claim 6.
8. host cell, it can express the described protein of claim 1.
9. connect the method for phosphodiester bond, comprising:
(a) select ligase enzyme according to claim 1 and 2;
(b) described ligase enzyme is mixed with DNA, described DNA contains the point of contact at least one chain of described DNA; With
(c) connect described phosphodiester bond at described point of contact.
10. method according to claim 9, wherein said ligase enzyme are the thermally-stabilised ligase enzymes that obtains from the archeobacteria isolate.
11. method according to claim 9, wherein said archeobacteria isolate are certain (Thermococcus sp.) (strain 9 deg n-7) of thermophilic Coccus.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US71729605P | 2005-09-15 | 2005-09-15 | |
| US60/717,296 | 2005-09-15 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN101287828A true CN101287828A (en) | 2008-10-15 |
Family
ID=37546966
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNA2006800383873A Pending CN101287828A (en) | 2005-09-15 | 2006-09-15 | Discovery, cloning and purification of thermococcus sp. (strain 9 deg n-7) DNA ligase |
Country Status (5)
| Country | Link |
|---|---|
| US (1) | US20090142811A1 (en) |
| EP (1) | EP1929000A1 (en) |
| JP (1) | JP2009508488A (en) |
| CN (1) | CN101287828A (en) |
| WO (1) | WO2007035439A1 (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109477127A (en) * | 2016-03-14 | 2019-03-15 | R基因股份有限公司 | Uht-stable lysine-saltant type ssDNA/RNA ligase |
| CN113481174A (en) * | 2021-07-01 | 2021-10-08 | 温州医科大学 | Nucleic acid ligase |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20090246788A1 (en) | 2008-04-01 | 2009-10-01 | Roche Nimblegen, Inc. | Methods and Assays for Capture of Nucleic Acids |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5494810A (en) * | 1990-05-03 | 1996-02-27 | Cornell Research Foundation, Inc. | Thermostable ligase-mediated DNA amplifications system for the detection of genetic disease |
| US5700672A (en) * | 1992-07-23 | 1997-12-23 | Stratagene | Purified thermostable pyrococcus furiousus DNA ligase |
| JP2000308494A (en) * | 1999-02-26 | 2000-11-07 | Tadayuki Imanaka | Hyperthermostable dna ligase |
-
2006
- 2006-09-15 WO PCT/US2006/035919 patent/WO2007035439A1/en active Application Filing
- 2006-09-15 EP EP06803634A patent/EP1929000A1/en not_active Withdrawn
- 2006-09-15 US US12/067,000 patent/US20090142811A1/en not_active Abandoned
- 2006-09-15 JP JP2008531333A patent/JP2009508488A/en not_active Withdrawn
- 2006-09-15 CN CNA2006800383873A patent/CN101287828A/en active Pending
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109477127A (en) * | 2016-03-14 | 2019-03-15 | R基因股份有限公司 | Uht-stable lysine-saltant type ssDNA/RNA ligase |
| CN113481174A (en) * | 2021-07-01 | 2021-10-08 | 温州医科大学 | Nucleic acid ligase |
| CN113481174B (en) * | 2021-07-01 | 2022-08-19 | 温州医科大学 | Nucleic acid ligase |
Also Published As
| Publication number | Publication date |
|---|---|
| US20090142811A1 (en) | 2009-06-04 |
| WO2007035439A8 (en) | 2008-05-02 |
| WO2007035439A1 (en) | 2007-03-29 |
| EP1929000A1 (en) | 2008-06-11 |
| JP2009508488A (en) | 2009-03-05 |
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Open date: 20081015 |